Geroprotectors: A role in the treatment of frailty.

The proportion of the population over the age of 65 is growing the most rapidly due to the longevity revolution. Frailty is prevalent in this age group and strongly associated with disability and hospitalization, having a significant impact on the costs of health and social care. New effective interventions to delay or reverse frailty are urgently required. Geroprotectors are a new class of drugs, which target fundamental mechanisms of ageing and show promise in delaying the onset of or boosting resilience in frail older people. However, there are challenges to their clinical translation. Here we review the literature for evidence that frailty can be delayed or reversed and geroprotectors can improve frailty in murine models and in patients. We will then discuss the challenges, which make their clinical testing complex and propose potential options for moving forward.

Stimulation frequency-noradrenaline release relationships examined in alpha2A-, alpha2B- and alpha2C-adrenoceptor-deficient mice.

The stimulation frequency-noradrenaline release relationship was studied in the vas deferens and the cerebral cortex of NMRI mice, mice in which the alpha2A-, the alpha2B-, the alpha2C- or both the alphaCA- and the alpha2C-adrenoceptor gene had been disrupted (alpha2AKO, alpha2BKO, alpha2CKO and alpha2ACKO), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically with a constant number of pulses (30 in vas deferens and 50 in brain cortex) at frequencies between 0.03 and 100 Hz. The frequency-evoked tritium overflow curves ascended monophasically in the vas deferens of wildtype and NMRI mice. Disruption of the alpha2B-adrenoceptor gene caused no change. In the vas deferens of alpha2CKO mice, the overflow evoked by low frequencies (0.3 and 1 Hz) was slightly increased. In the vas deferens of alpha2AKO and alpha2ACKO mice, the evoked overflow was increased to a greater extent. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the vas deferens of NMRI, wildtype, alpha2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the vas deferens of alpha2AKO and alphaC2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine slightly steepened the slope of the vas deferens frequency-overflow curve. In the brain cortex of wildtype and NMRI mice, the frequency-evoked tritium overflow curves were U-shaped. In the brain cortex of alpha2BKO and alpha2CKO mice, the evoked overflow was slightly reduced. In the brain cortex of alpha2AKO and alpha2AcKO mice, in contrast, the evoked overflow was increased. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the brain cortex of NMRI, wildtype, Q2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the brain cortex of alpha2AKO and alpha2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine flattened the U shape of the brain cortex frequency-overflow curve. It is concluded that alpha2-autoinhibition is one factor that shapes the frequency-noradrenaline release relationships in the mouse vas deferens and cerebral cortex. The autoreceptors are mainly alpha2A and to a minor extent, and well detectable in the vas deferens only, alpha2C. When both the alpha2A- and the alpha2C-adrenoceptor have been deleted, alpha2B-adrenoceptors may be expressed as autoreceptors in noradrenergic neurons. It seems possible that alpha2C-autoreceptors depress mainly release at low (around 1 Hz) whereas alpha2A-autoreceptors depress mainly release at high (around 10 Hz) frequencies.

Postnatal development of presynaptic receptors that modulate noradrenaline release in mice.

The objective of the study was to clarify the postnatal development of the following transmitter release-modulating receptors of noradrenergic neurons in mice: alpha2-adrenoceptors, muscarinic, opioid and cannabinoid receptors (inhibitory), beta-adrenoceptors and receptors for angiotensin II and bradykinin (facilitatory). Wildtype (NMRI) and in some cases alpha2A/D-adrenoceptor-deficient mice aged 1 day (P1) or 8-16 weeks (adults) were used. Hippocampal and occipito-parietal cortex slices and sympathetically innervated tissues (atria and vas deferens) were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation led to distinct increases in tritium efflux which were abolished by tetrodotoxin or removal of calcium. Concentration-response curves of appropriate agonists and in the case of alpha2-autoreceptors antagonists were determined. For beta-adrenoceptors and angiotensin receptors, the interaction of agonists with antagonists was also examined. Results demonstrate that alpha2A/D-autoreceptors operate already at P1 whereas nonalpha2A/D-autoreceptors, presumably alpha2C, develop later. Of the various heteroreceptors, those of brain noradrenergic neurons (OP3 and ORL1) modulate the release of [3H]-noradrenaline at least as effectively at P1 as in adults. Those of peripheral sympathetic neurons (muscarinic, probably mainly M2, OP1, OP2, OP3, CB1, AT1 and B1), in contrast, operate less effectively or not at all at P1, with one exception: beta2-adrenoceptors increase the release of [3H]-noradrenaline (atria) to the same extent, irrespective of age. Overall, results indicate that brain and peripheral noradrenergic neurons release their transmitter already shortly after birth. Presynaptic receptor mechanisms mature differentially in the brain and the periphery. Moreover, the various presynaptic receptors differ in their postnatal development and may play differential roles at different ages.

A study of presynaptic alpha2-autoreceptors in alpha2A/D-, alpha2B- and alpha2C-adrenoceptor-deficient mice.

The function of presynaptic alpha2-autoreceptors was studied in the hippocampus, occipito-parietal cortex, atria and vas deferens of NMRI mice, mice in which the alpha2A/D-, the alpha2B- or alpha2c-adrenoceptor gene had been disrupted (alpha2A/DKO, alpha2BKO and alpha2CKO, respectively), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The alpha2-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium in all tissues from all mouse strains (stimulation with single pulses or single high-frequency pulse trains, called POPs, i.e. pulse patterns leading to minimal autoinhibition). The effects of medetomidine did not differ in NMRI, wildtype, alpha2BKO and alpha2CKO mice but were greatly reduced in alpha2A/DKO brain preparations and to a lesser extent in alpha2A/DKO atria and vasa deferentia. Six drugs were tested as antagonists against medetomidine. Their pKd values indicated that the hippocampal and occipito-parietal alpha2-autoreceptors in NMRI and wildtype mice were alpha2D (the rodent variant of the alpha2A/D-adrenoceptor) whereas the atrial and vas deferens alpha2-autoreceptors in NMRI and wildtype mice could not be identified with a single alpha2 subtype. Deletion of the alpha2A/D gene changed the pKd values in all tissues so that they now reflected alpha2C properties, whereas deletion of the alpha2C gene changed the pKd values in atria and vasa deferentia so that they now had alpha2D properties (as they had in NMRI and wildtype brain preparations). Autoinhibition by released noradrenaline was created using trains of up to 64 pulses or up to 4 POPs, and the overflow-enhancing effect of the alpha2 antagonist rauwolscine was determined. Results did not differ, irrespective of whether preparations were obtained from NMRI, wildtype, alpha2BKO or alpha2CKO mice: the overflow of tritium elicited by p pulses or POPs was much smaller than p times the overflow elicited by a single pulse or POP, and rauwolscine greatly increased the evoked overflow. Results differed, however, in tissues taken from alpha2A/DKO mice: in these tissues, the overflow of tritium elicited by p pulses or POPs was close to p times the overflow elicited by a single pulse or POP, and rauwolscine did not increase the evoked overflow of tritiumor increased it only marginally. When a greater degree of autoinhibition was produced in atria and vasa deferentia by stimulation with 120 pulses, both disruption of the alpha2A/D gene and disruption of the alpha2C gene but not disruption of the alpha2B gene attenuated the overflow-enhancing effects of phentolamine and rauwolscine. In NMRI and wildtype atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine at enhancing the evoked overflow were not easily compatible with a single alpha2 subtype. In alpha2A/DKO atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine indicated that the autoinhibition-mediating receptors were alpha2C, whereas in alpha2CKO atria and vasa deferentia the relative potencies indicated that the autoinhibition-mediating receptors were alpha2D. It is concluded that alpha2-autoreceptors function identically in NMRI mice and the wildtype mice from which the receptor-deficient animals had been generated. There is no evidence from the experiments for any contribution of alpha2B-adrenoceptors to autoreceptor function. The main presynaptic alpha2-autoreceptors are alpha2A/D, both as sites of action of exogenous agonists and as sites of action of previously released noradrenaline. However, there are in addition non-alpha2A/D-, probably alpha2C-autoreceptors. They are less prominent in mediating the inhibitory effects of exogenous agonists and the negative feedback effect of released noradrenaline. They operate not only after deletion of the alpha2A/D-adrenoceptors but also in normal (NMRI, wildtype) mice without gene deletion.

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides.

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.

Alpha2-adrenoceptor-mediated inhibition of cultured sympathetic neurons: changes in alpha2A/D-adrenoceptor-deficient mice.

Alpha2-Adrenoceptor-mediated inhibition of [3H]noradrenaline release and alpha2-adrenoceptor-mediated inhibition of voltage-activated Ca2+ currents were compared in cultured thoracolumbar postganglionic sympathetic neurons from newborn wildtype (WT) mice and mice in which the alpha2A/D-adrenoceptor gene had been disrupted (alpha2A/DKO). In cultures prepared from WT mice and preincubated with [3H]noradrenaline, the alpha2-adrenoceptor agonist 5-bromo-6-(2-imidazolidinylidenamino)quinoxaline (UK 14,304) reduced the (autoinhibition-free) release of [3H]noradrenaline elicited by single electrical pulses or trains of 8 pulses at 100 Hz. The maximal inhibition by UK 14,304 amounted to 70%-85%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) and, to a smaller extent, rauwolscine (0.3 microM). Pretreatment of the cultures with pertussis toxin abolished the effect of UK 14,304. Phentolamine and rauwolscine increased the (alpha2-autoinhibited) release of [3H]noradrenaline elicited by 18, 36 or 72 pulses at 3 Hz. In cultures from alpha2A/DKO mice, UK 14,304 failed to reduce the release of [3H]noradrenaline elicited by single pulses and phentolamine and rauwolscine failed to increase the release of [3H]noradrenaline elicited by 18-72 pulses at 3 Hz. In neurons from WT mice examined with the amphotericin B-perforated configuration of the patch clamp method, UK 14,304 reduced depolarisation-evoked Ca2+ currents. The inhibition was voltage-dependent as shown by a decline at strong depolarisation during ramp-like voltage commands and by an attenuation briefly after a conditioning depolarising pulse. The maximal inhibition by UK 14,304 was 39%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) but not significantly changed by rauwolscine (0.3 microM) and prazosin (1 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304. In neurons from alpha2A/DKO mice, UK 14,304 also reduced depolarisation-evoked Ca2+ currents, but with a smaller maximal effect, namely 18% inhibition. Its concentration-response curve was shifted to the right by rauwolscine (0.3 microM) and prazosin (1 microM) but not significantly changed by phentolamine (0.3 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304 also in cultures from alpha2A/DKO mice. It is concluded that the only presynaptic alpha2-autoreceptors that detectably depress transmitter release from cultured thoracolumbar sympathetic neurons taken from newborn mice are alpha2A/D. In contrast, the soma-dendritic alpha2-autoreceptors that inhibit voltage-gated Ca2+ channels are both alpha2A/D and non-alpha2A/D (i.e. alpha2B or alpha2c). Both presynaptic alpha2A/D- and soma-dendritic alpha2A/D- and non-alpha2A/D-autoreceptors operate through pertussis toxin-sensitive G proteins in these neurons.

Alpha2-adrenoceptors modulating neuronal serotonin release: a study in alpha2-adrenoceptor subtype-deficient mice.

1. The release-inhibiting alpha2-adrenoceptors of cerebral serotoninergic axons were studied in mice. Slices of the hippocampus or the occipito-parietal cortex from NMRI mice, from mice lacking the alpha2A/D-, the alpha2B-, the alpha2C- or both the alpha2A/D- and the alpha2C-adrenoceptor, and from mice sharing the genetic background of the receptor-deficient animals (WT) were preincubated with [3H]-serotonin and then superfused and stimulated electrically, in most experiments by trains of 8 pulses at 100 Hz. 2. The concentration-response curves of the alpha2-adrenoceptor agonist medetomidine were virtually identical in hippocampal slices from NMRI and WT mice, with maximally 70% inhibition and an EC50 of about 2 nM. In hippocampal slices from NMRI mice, phentolamine and rauwolscine were equipotent antagonists against medetomidine. 3. The effect of medetomidine was greatly reduced, with maximally 20% inhibition, in hippocampal slices from alpha2A/D-adrenoceptor-deficient mice; was slightly reduced, with maximally 59% inhibition, in hippocampal slices from alpha2C-adrenoceptor-deficient mice; was not changed in hippocampal slices from alpha2B-adrenoceptor-deficient mice; and was abolished in hippocampal slices from mice lacking both the alpha2A/D- and the alpha2C-adrenoceptor. 3. Similar results were obtained in: (i) occipito-parietal slices from NMRI and alpha2A/D-adrenoceptor-deficient mice and (ii) hippocampal slices that were preincubated with [3H]-serotonin in the presence of oxaprotiline to rule out cross-labelling of noradrenergic axons. 5. The serotoninergic axons of the mouse brain possess both alpha2A/D-heteroreceptors, which predominate, and alpha2C-heteroreceptors but lack alpha2B-adrenoceptors. The situation resembles the coexistence of alpha2A/D- and alpha2C-autoreceptors but lack of alpha2B-autoreceptors at the noradrenergic axons of mice.

Electrically evoked release of [(3)H]noradrenaline from mouse cultured sympathetic neurons: release-modulating heteroreceptors.

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting alpha(2)-autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [(3)H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M(2) muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB(1) receptors. Prostaglandin E(2), sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E(2), and somatostatin were abolished (at the highest concentration of WIN 55, 212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the beta(2)-adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.

Modulation of (3)H-noradrenaline release by presynaptic opioid, cannabinoid and bradykinin receptors and beta-adrenoceptors in mouse tissues.

Release-modulating opioid and cannabinoid (CB) receptors, beta-adrenoceptors and bradykinin receptors at noradrenergic axons were studied in mouse tissues (occipito-parietal cortex, heart atria, vas deferens and spleen) preincubated with (3)H-noradrenaline. Experiments using the OP(1) receptor-selective agonists DPDPE and DSLET, the OP(2)-selective agonists U50488H and U69593, the OP(3)-selective agonist DAMGO, the ORL(1) receptor-selective agonist nociceptin, and a number of selective antagonists showed that the noradrenergic axons innervating the occipito-parietal cortex possess release-inhibiting OP(3) and ORL(1) receptors, those innervating atria OP(1), ORL(1) and possibly OP(3) receptors, and those innervating the vas deferens all four opioid receptor types. Experiments using the non-selective CB agonists WIN 55,212-2 and CP 55,940 and the CB(1)-selective antagonist SR 141716A indicated that the noradrenergic axons of the vas deferens possess release-inhibiting CB(1) receptors. Presynaptic CB receptors were not found in the occipito-parietal cortex, in atria or in the spleen. Experiments using the non-selective beta-adrenoceptor agonist isoprenaline and the beta(2)-selective agonist salbutamol, as well as subtype-selective antagonists, demonstrated the occurrence of release-enhancing beta(2)-adrenoceptors at the sympathetic axons of atria and the spleen, but demonstrated their absence in the occipito-parietal cortex and the vas deferens. Experiments with bradykinin and the B(2)-selective antagonist Hoe 140 showed the operation of release-enhancing B(2) receptors at the sympathetic axons of atria, the vas deferens and the spleen, but showed their absence in the occipito-parietal cortex. The experiments document a number of new presynaptic receptor locations. They confirm and extend the existence of marked tissue and species differences in presynaptic receptors at noradrenergic neurons.

Enhancement of noradrenaline release by angiotensin II and bradykinin in mouse atria: evidence for cross-talk between G(q/11) protein- and G(i/o) protein-coupled receptors.

1. The interaction between alpha(2)-autoreceptors and receptors for angiotensin (AT(1)) and bradykinin (B(2)) was studied in mouse isolated atria. The preparations were labelled with [(3)H]-noradrenaline and then superfused with desipramine-containing medium and stimulated electrically. 2. Angiotensin II (10(-11) - 10(-7) M), angiotensin III (10(-10) - 10(-6) M) and bradykinin (10(-11) - 10(-7) M) enhanced the evoked overflow of tritium when preparations were stimulated with conditions that led to marked alpha(2)-autoinhibition (120 pulses at 3 Hz), but not when stimulated with conditions that led to little alpha(2)-autoinhibition (20 pulses at 50 Hz). 3. Blockade of alpha-adrenoceptors by phentolamine (1 or 10 microM) reduced or abolished the effect of angiotensin II and bradykinin on the overflow response to 120 pulses at 3 Hz. 4. Addition of the delta-opioid agonist [D-Ser(2)]-leucine enkephalin-Thr (DSLET, 0.1 microM), or of neuropeptide Y (0.1 microM), together with phentolamine, restored the effect of angiotensin II and bradykinin. 5. The beta-adrenoceptor agonist terbutaline (10(-9) - 10(-4) M) enhanced the evoked overflow of tritium irrespective of the degree of autoinhibition. 6. The experiments show that (i) a marked prejunctional facilitatory effect of angiotensin and bradykinin in mouse isolated atria requires prejunctional alpha(2)-autoinhibition; (ii) in the absence of alpha(2)-autoinhibition, activation of other prejunctional G(i/o) protein-coupled receptors, namely opioid and neuropeptide Y receptors, restores a marked effect of angiotensin II and bradykinin; and (iii) the facilitatory effect of terbutaline is not dependent upon the degree of alpha(2)-autoinhibition. The findings indicate that the major part of the release-enhancing effect elicited through prejunctional G(q/11) protein-coupled receptors is due to disruption of an ongoing, alpha(2)-autoreceptor-triggered G(i/o) protein mediated inhibition.

P2 receptor-mediated inhibition of dopamine release in rat neostriatum.

Axon terminal nucleotide P2 receptors mediating an inhibition of transmitter release have, so far, been detected in various sympathetically innervated tissues,(8,27) and on central noradrenergic,(14,26) glutamatergic(15) and serotonergic neurons. (28) We have now investigated the effect of ATP and related nucleotides on the release of endogenous dopamine from slices of rat neostriatum using fast cyclic voltammetry. Mutual interactions between the two neurotransmitters have been observed previously: ATP and related nucleotides induce a release of dopamine in PC12 pheochromocytoma cells, a frequently used model for sympathetic neurons;(10,22) they also increase the dopamine concentration in rat brain measured by in vivo microdialysis(16,32) and stimulate the uptake of dopamine by rat striatal synaptosomes.(3) Dopamine, in contrast, facilitates activation of ligand-gated cation channels (i. e. P2X(2) receptors) by ATP.(11,20) Here, we show that ATP and two of its analogues decrease the electrically evoked release of endogenous dopamine in rat neostriatum. The inhibitory effect of ATP is blocked by the P2 receptor antagonists suramin, reactive blue 2 and cibacron blue 3GA. Suramin, in addition, partly prevents the attenuation of dopamine release evoked by a single stimulus that follows a brief train of high-frequency pulses.These findings suggest the existence of release-inhibiting P2 receptors on dopaminergic nerve terminals and indicate that dopaminergic transmission in rat neostriatum might be modulated by an endogenous P2 receptor ligand, presumably ATP.

Occurrence, pharmacology and function of presynaptic alpha2-autoreceptors in alpha2A/D-adrenoceptor-deficient mice.

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.

Mouse postganglionic sympathetic neurons: primary culturing and noradrenaline release.

Basic properties of noradrenaline release were studied in primary cultures of thoracolumbar postganglionic sympathetic neurons taken from 1-3-day-old NMRI mice. After 7 days in vitro, the cultures were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Conventional trains of pulses (for example, 36 pulses at 3 Hz) as well as single pulses and brief high-frequency trains (for example, four pulses at 100 Hz) elicited a well-measurable overflow of tritium, which was abolished by 0.3 microM tetrodotoxin or omission of Ca2+, but not changed by 1 microM rauwolscine. In trains of one, two, four, six, eight, or 10 pulses at 3 Hz, the evoked overflow of tritium remained constant from pulse to pulse at 1.3 mM Ca2+, but declined slightly at 2.5 mM Ca2+. Tetraethylammonium at 10 mM selectively increased the overflow elicited by small pulse numbers and especially by a single pulse. In trains of 10 pulses delivered at 0.3, 1, 3, 10, 30, or 100 Hz, the evoked overflow of tritium increased from 0.3 to 30 Hz and then declined at 100 Hz. This relationship was particularly pronounced at low Ca2+ concentrations (for example, 0.3 mM). Tetraethylammonium at 10 mM selectively increased the overflow elicited by low frequencies of stimulation. It is concluded that primary cultures of mouse postganglionic sympathetic neurons can be used to investigate release of [3H]noradrenaline. The release is well measurable, even upon a single electrical pulse. It agrees with release in intact sympathetically innervated tissues in a number of fundamental properties, including the pulse number and frequency dependence. The preparation may be of special interest in conjunction with genetic manipulations in the donor animals.

Noradrenaline release from cultured mouse postganglionic sympathetic neurons: autoreceptor-mediated modulation.

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.

Prejunctional angiotensin receptors involved in the facilitation of noradrenaline release in mouse tissues.

The effect of angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) on the electrically induced release of noradrenaline was studied in preparations of mouse atria, spleen, hippocampus, occipito-parietal cortex and hypothalamus preincubated with [3H]-noradrenaline. The prejunctional angiotensin receptor type was investigated using the non-selective receptor antagonist saralasin (AT1/AT2) and the AT1 and AT2 selective receptor antagonists losartan and PD 123319, respectively. In atrial and splenic preparations, angiotensin II (0.01 nM-0.1 microM) and angiotensin III (0.01 and 0.1 nM-1 microM) increased the stimulation-induced overflow of tritium in a concentration-dependent manner. Angiotensin IV, only at high concentrations (1 and 10 pM), enhanced tritium overflow in the atria, while angiotensin-(1-7) (0.1 nM-10 microM) was without effect in both preparations. In preparations of hippocampus, occipito-parietal cortex and hypothalamus, none of the angiotensin peptides altered the evoked overflow of tritium. In atrial and splenic preparations, saralasin (0.1 microM) and losartan (0.1 and 1 microM), but not PD 123319 (0.1 microM), shifted the concentration-response curves of angiotensin II and angiotensin III to the right. In conclusion, in mouse atria and spleen, angiotensin II and angiotensin III facilitate the action potential induced release of noradrenaline via a prejunctional AT1 receptor. Only high concentrations of angiotensin IV are effective in the atria and angiotensin-(1-7) is without effect in both preparations. In mouse brain areas, angiotensin II, angiotensin III, angiotensin IV and angiotensin-(1-7) do not modulate the release of noradrenaline.

Abnormal regulation of the sympathetic nervous system in alpha2A-adrenergic receptor knockout mice.

alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.

A search for presynaptic imidazoline receptors at rabbit and rat noradrenergic neurones in the absence of alpha 2-autoinhibition.

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing alpha 2-autoinhibition. We tried to find them under alpha2-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through alpha 2-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (alpha 2-autoinhibition-free conditions), all ten "agonists" [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), alpha-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten "agonists" with similar potency, the pKd-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pKd 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pKd-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pKd of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked alpha 2-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical alpha 2-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of alpha 2-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pKd-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the alpha 2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an alpha 2(A)-autoreceptor agonist in rabbit tissues but as an alpha 2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) alpha 2A-adrenoceptors than the orthologous (rodent) alpha 2D-adrenoceptors.

Presynaptic alpha 2A/D-autoreceptors in the brain cortex of Cercopithecus aethiops.

The existence of presynaptic alpha 2-autoreceptors, and the subtype to which the receptors belong, were studied in the brain cortex from two African green monkeys (Cercopithecus aethiops). Slices of the brain cortex were preincubated with 3H-noradrenaline. The slices were then superfused with medium containing desipramine (1 microM) and stimulated electrically with trains of 72 pulses. 3 Hz. Concentration-response curves of nine alpha-adrenoceptor antagonists were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, i.e. the release of noradrenaline, indicating the existence of presynaptic alpha 2-autoreceptors. The EC30%, values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity for the auto-receptors. The subclassification was based on a comparison of these affinity estimates with affinities for alpha 2A, alpha 2B, alpha 2C and alpha 2D radioligand binding sites and for previously classified alpha 2A- and alpha 2D-autoreceptors. The comparison indicates that the alpha 2-autoreceptors in the brain cortex of Cercopithecus aethiops belong to the genetic alpha 2A/D and not the alpha 2B or alpha 2C subtype. In their pharmacological properties, the autoreceptors are closest to the pharmacological alpha 2D subtype. The results support the notion that alpha 2-autoreceptors belong at least predominantly to the alpha 2A/D subtype of alpha 2-adrenoceptors.

A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra.

It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.

The presynaptic alpha-2 autoreceptors in pig brain cortex are alpha-2A.

The presynaptic alpha-2 autoreceptors in pig brain cortex were subclassified in terms of alpha-2A, alpha-2B, alpha-2C and alpha-2D to test the hypothesis that alpha-2 autoreceptors belong predominantly to the alpha-2A/D pair of orthologous alpha-2 adrenoceptors. Slices of brain cortex were preincubated with [3H]norepinephrine and then superfused and stimulated electrically. pKd values of 13 alpha-2 adrenoceptor antagonists (including the partial agonist oxymetazoline) against the alpha-2 agoinst 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) were determined. The stimulation periods used (six pulses at 100 Hz) did not lead to alpha-2 autoinhibition, as shown by the failure of all but one of the alpha-2 antagonists to increase the stimulation-evoked overflow of tritium. UK 14,304 caused a concentration-dependent decrease of the evoked overflow of tritium, with an EC50 value of 0.90 nM and a maximal inhibition of 95.2%. All antagonists shifted the concentration-inhibition curve of UK 14,304 to the right in a parallel manner. Antagonist pKd values were calculated from the shifts. The pKd values at the presynaptic alpha-2 autoreceptors in pig brain cortex correlated excellently with pKd values at previously subclassified alpha-2A sites but did not correlate significantly or correlated much less well with pKd values at alpha-2B, alpha-2C and alpha-2D sites. Also, ratios of Kd values of the antagonists at the presynaptic alpha-2 autoreceptors in pig brain cortex agreed well with ratios at previously subclassified alpha-2A sites but not with those at previously subclassified alpha-2B-D sites. It is concluded that the alpha-2 autoreceptors in pig brain cortex are alpha-2A, in accordance with the hypothesis mentioned.