Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings.

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (P(o)) of the calcium release channel at activating calcium concentrations (20-100 microM Ca(2+)) by 60-100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6-2 mM) decreased P(o). Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced P(o) at activating Ca(2+) (20-100 microM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca(2+) (0.6 microM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca(2+). High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca(2+). The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.

Amphetamine reverses or blocks the operation of the human noradrenaline transporter depending on its concentration: superfusion studies on transfected cells.

Whether amphetamine enhances noradrenergic activity by uptake blockade or a releasing action is still a matter of debate. In order to gain insight into the interaction of amphetamine with the noradrenaline transporter its cDNA was transfected into COS-7 cells (NAT-cells) or cotransfected with the cDNA of the vesicular monoamine transporter (NAT/VMAT-cells); cells were loaded with [3H]noradrenaline, superfused and the efflux analysed for total tritium and [3H]noradrenaline. In NAT-cells amphetamine stimulated [3H]noradrenaline efflux concentration-dependently when added to the superfusion buffer at 0.01, 0.1 and 1 microM. By contrast, 10 or 100 microM amphetamine stimulated efflux to a smaller extent or not at all; however, on switching back to amphetamine-free buffer a prompt increase of efflux was observed. Cocaine did not increase efflux per se and blocked the amphetamine-induced efflux. In NAT/VMAT-cells amphetamine stimulated efflux in a concentration-dependent manner. The effect showed saturation at 1 microM and was not suppressed at higher concentrations. Cocaine also elicited efflux from NAT/VMAT-cells concentration-dependently; the maximum was reached at approximately 1 microM and amounted to only about half of the amphetamine-induced efflux. It is concluded that amphetamine can induce noradrenaline transporter mediated release only at high nanomolar to low micromolar concentrations. At higher concentrations it blocks the noradrenaline transporter; in this case, the releasing action of amphetamine, like that of cocaine, is dependent on a vesicular pool of noradrenaline.

Central alpha 2-autoreceptors: agonist dissociation constants and recovery after irreversible inactivation.

1. Rats received an intraperitoneal injection of 1.6 mg kg-1 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of presynaptic release-modulating alpha 2-autoreceptors. Cerebral cortex slices were prepared at different times after the injection (24, 48, 96, 192, 336, 774 h), incubated with [3H]-noradrenaline ([3H]-NA), superfused and stimulated electrically with 4 pulses at 100 Hz (= autoinhibition-free condition). Overflow of radioactivity was used to measure release. Furchgott analysis was used to estimate agonist dissociation constants (KA) and pool size of resynthesized receptors (q). 2. The KA values of the three alpha 2-autoreceptor agonists, bromoxidine (UK-14304), clonidine and noradrenaline (NA) were 187 nM, 72 nM, and 1202 nM, respectively. 3. The release-inhibiting effects of the agonists returned considerably faster than the receptor pool. The calculated half-lives for the recovery of the maximal release-inhibiting effects of bromoxidine, clonidine and NA were 30.7, 63.6 and 20.8 h, respectively, whereas the half-life for the recovery of the receptor pool was 445 h. 4. The data indicate a large receptor reserve at presynaptic alpha 2-autoreceptors for the agonists used and validate the use of EEDQ as a tool for the determination of agonist dissociation constants.

Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

Rapid, agonist-induced desensitization of alpha 2-autoreceptors modulating transmitter release.

1. The release of previously incorporated [3H]-noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 mumol l-1 of the uptake inhibitor, cocaine, or the selective alpha 2-adrenoceptor agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 4,304), was applied for different periods of time in order to detect a possible time-dependence of the alpha 2-adrenoceptor-mediated inhibition of electrically evoked tritium outflow. 2. In chick sympathetic neurones, stimulation-evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 mumol l-1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 mumol l-1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l-1 to 10 mumol l-1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations > or = 10 nmol l-1. 3. In rat sympathetic neurones, noradrenaline (100 nmol l-1) reduced stimulation-evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 mumol l-1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK14,304 for either 2 or 16 min did not result in significantly different IC5o values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations > 10 nmol 1-1.4. In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 micromol 1-1 UK14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.5 The results demonstrate that alpha 2-adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist-induced desensitization within minutes. Such rapid desensitization of alpha 2-autoreceptors was not detected in brain slice preparations.

Evidence against a functional link between noradrenaline uptake mechanisms and presynaptic alpha-2 adrenoceptors.

Slices prepared from rat cerebral cortex were labelled with 3H-noradrenaline and superfused. Electrical field stimulation was carried out 15 min (S1) and 45 min (S2) after the start of collection of 5-min samples using 4 pulses delivered at 100 Hz. Drugs acting at alpha 2-adrenoceptors were added 20 min before S2, and their effects were evaluated using the S2/S1-ratio. The alpha 2-adrenoceptor antagonists idazoxan (1 mumol/l) and rauwolscine (1 mumol/l) failed to increase stimulation-evoked overflow of radioactivity in the absence or presence of the noradrenaline reuptake inhibitor desipramine (1 mumol/l). This indicates that the duration of electrical stimulation was too short to allow development of alpha 2-adrenoceptor-mediated autoinhibition by released noradrenaline. The effect of clonidine (3-1000 nmol/l) on stimulation-evoked overflow of radioactivity was tested in the absence and presence of three different reuptake inhibitors (desipramine, 1 mumol/l; maprotiline, 1 mumol/l; cocaine, 10 mumol/l). The analysis yielded identical concentration-response curves under all conditions. These results argue against an action of inhibitors of neuronal reuptake of noradrenaline at the presynaptic alpha 2-adrenoceptor and against the concept of a functional link between uptake site and receptor.

Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic alpha-adrenoceptors and lack of autoinhibition.

Sympathetic neurons from twelve day old chick embryos were plated on polystyrol discs and kept in culture for five days. After incubation with 3H-noradrenaline the discs were transferred to small chambers and superfused. Electrical field stimulation (36 pulses at 3 Hz) increased the outflow of tritium. The evoked overflow of tritium was abolished in the absence of extracellular calcium and was diminished by about 90% in the presence of tetrodotoxin (1 mumol/l). The alpha 2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the alpha 1-adrenoceptor agonist methoxamine was ineffective at up to 1 mumol/l. The concentration-response curve of UK-14,304 was shifted to the right by the alpha 2-adrenoceptor antagonist yohimbine (0.03 mumol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 mumol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating alpha 2-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition.

Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via alpha 2-adrenoceptors in chick sympathetic neurons.

Effects of alpha 2-adrenoceptor agonists on whole-cell Ca2+ currents and 3H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the alpha 2-adrenoceptor-mediated inhibition of Ca2+ currents and the modulation of noradrenaline release caused by noradrenaline (1 mumol/l; in the presence of 10 mumol/l cocaine) or the alpha 2-adrenoceptor agonists 5-bromo-6-(2-imidazolin-2- ylamino)quinoxaline (UK 14,304, 10 mumol/l) and clonidine (10 mumol/l). These results suggest that the alpha 2-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca2+ channels via pertussis-toxin-sensitive GTP-binding-proteins.

[Central presynaptic receptors]. / Zentrale präsynaptische Rezeptoren.

Experiments on two different inhibitory presynaptic receptor systems are presented. 1. Superfused and electrically stimulated brain slices are a widely used experimental model to study the release of noradrenaline and its modulation by inhibitory alpha-2 adrenoceptors. By using a minisuperfusion chamber we succeeded in studying the simplest case of autoinhibition, i.e. the release of transmitter induced by a single pulse and two consecutive pulses, respectively. When electrical stimulation is performed using a single pulse, no autoinhibition is possible, whereas following stimulation with two pulses the transmitter released by the first pulse will inhibit the effect of the second pulse. By systemically varying the time interval between the two pulses the minimal time requirement for development of autoinhibition was determined to be 100 ms. Short pulse trains of high frequency such as 4 pulses within 30 ms circumvent autoinhibition and cause inhibition-free release by each applied pulse. The release of transmitter evoked in this way is not only free from autoinhibition but, in addition, easily measurable, which makes this method of stimulation very suitable for analyses at presynaptic receptors. By using this approach it became possible, for the first time, to determine dissociation constants of antagonists and agonists at the central presynaptic alpha-2 adrenoceptor without the distortion introduced by autoinhibition occurring during release. 2. There is a substantial body of evidence for a role of medullary serotonergic nerve cells in the regulation of blood pressure and heart rate. It is hypothesized that the serotonergic neurons project to the thoracic spinal cord exerting a tonic excitatory influence on presynaptic sympathetic neurons of the intermediolateral cell column. Experiments were performed in pentobarbital anaesthetized rats to reduce this excitatory tone by activating inhibitory autoreceptors which are located on the perikarya and dendrites on the serotonergic cells and which have been shown to belong to the 5-HT1A subtype. Local stereotactic injection of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) caused a decrease in mean arterial blood pressure (MAP) and heart rate (HR). The effects were blocked by pretreatment of the animals with the 5-HT1A antagonist spiroxatrine. Moreover, neurochemical lesioning of serotonergic neurons by intracisternal injection of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) abolished the effects of 8-OH-DPAT. Bilateral intraspinal injection of 5,7-DHT, which interrupts the medullo-spinal serotonergic pathway, markedly attenuated the effects of local intramedullary injection of 8-OH-DPAT.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of transmitter release from rat sympathetic neurons via presynaptic M(1) muscarinic acetylcholine receptors.

BACKGROUND AND PURPOSE: M(2), M(3) and/or M(4) muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M(1) receptors mediate an inhibition of K(v)7, Ca(V)1 and Ca(V)2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M(1) receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M(1) receptors. EXPERIMENTAL APPROACH: In primary cultures of rat superior cervical ganglion neurons, Ca(2+) currents were recorded via the perforated patch-clamp technique, and the release of [(3)H]-noradrenaline was determined. KEY RESULTS: The muscarinic agonist oxotremorine M (OxoM) transiently enhanced (3)H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M(1) receptors, by opening K(v)7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening K(v)7 channels, but was reduced by PTX and omega-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M(1) receptors or inhibition of phospholipase C. When [(3)H]-noradrenaline release was triggered independently of voltage-activated Ca(2+) channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca(2+) currents by OxoM was also reduced by omega-conotoxin and PTX and was abolished by M(1) antagonism in PTX-treated neurons. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that M(1), in addition to M(2), M(3) and M(4), receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.

[Studies on the incidence of diabetes mellitus in obese patients]. / Untersuchungen zur Diabetes-mellitus-Inzidenz bei Fettsüchtigen.

In a retrospective investigation 228 obese persons with healthy metabolism and diabetics (NIDD) were invited to a check-up 4-10 years after a reduction of weight which was performed during a clinical treatment in the 2nd Medical Clinic of Halle University. 95 patients did not comply with the invitation, 9 patients had died in the meantime. Among the 24 obese persons examined were 9 patients with NIDD. In other 19 patients a diabetic metabolic disturbance had become manifest in the meantime. 78 obese persons underwent a clinical examination, among them 9 NIDD. 60 patients had a normal and 18 a pathological carbohydrate tolerance. Of these 18 patients in 9 patients an up to now unknown diabetes mellitus could be proved. The obesity coincides with a relatively high rate of disturbed carbohydrate tolerance and a larger proportion of manifest diabetics compared with the normal population. In the own group of patients the morbidity of diabetes among the obese persons increases from 10.5 to 30% after on an average 7 years. Obese persons who had essentially exceeded their initial weight in the meantime and show further metabolic disturbances are particularly endangered.

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4'-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/- 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4-CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4'-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

Short- and long-term functional alterations of the skeletal muscle calcium release channel (Ryanodine receptor) by Suramin: apparent dissociation of single channel current recording and [3H]ryanodine binding.

The present study demonstrates the following characteristic suramin actions on the purified skeletal muscle calcium release channel in single-channel current recordings and [(3)H]ryanodine binding to HSR: 1) Suramin (0.3-0.9 mM) induced a concentration-dependent increase in the open probability (P(o) congruent with 0.9) at 20 to 100 microM Ca(2+) and an almost fully open channel at 1 mM Ca(2+) (P(o) = 0.95) with a marked shift to longer open states (tau(o)3/tau(o)4). Suramin increased the apparent calcium affinity to the activating high-affinity calcium binding sites and reduced the apparent magnesium affinity to the inhibitory low affinity Ca(2+)/Mg(2+) binding sites. 2) Channel activation by suramin and sulfhydryl oxidation was additive. 3) Suramin (0.9 mM) reversed the Ca-calmodulin-induced channel inhibition at 0.1 or 1 to 5 microM Ca-calmodulin. 4) The open probability of the suramin activated channel was almost completely inhibited by 10 mM Mg(2+) or Ca(2+) on short suramin exposure. Prolonged suramin exposure (30-60 min) resulted in a time-dependent, slow increase in P(o), with long open states of low frequency in the presence of 10 to 20 mM Mg(2+) or Ca(2+). 5) Magnesium induced inhibition of P(o) (IC(50) = 0.38 mM) and equilibrium [(3)H]ryanodine binding (IC(50) = 0.30 mM) agreed well in control channels, but were dissociated in the presence of 0.9 to 1.0 mM suramin (IC(50) = 0.82 mM versus 83 mM). [(3)H]ryanodine binding seemed to monitor predominantly the long-term alteration in channel function. 6) The multiple effects of suramin on channel function suggest an allosteric mechanism and no direct effects on binding of endogenous ligands involved in channel gating.

Presynaptic autoinhibition of central noradrenaline release in vitro: operational characteristics and effects of drugs acting at alpha-2 adrenoceptors in the presence of uptake inhibition.

Functional characteristics of autoinhibition of central noradrenaline release were studied in the presence of uptake inhibition. Slices of rat cerebral cortex were incubated with [3H]noradrenaline, superfused and field-stimulated with 1 to 16 monophasic rectangular pulses at frequencies of 0.02 to 40 Hz. 1) Substances acting at presynaptic alpha-2 adrenoceptors were identified as antagonists, agonists or partial agonists by comparing their effects on 3H-overflow evoked by a single pulse or by two consecutive pulses at 1 Hz. 2) When 1 to 16 pulses were delivered at 0.02, 0.08, 0.3 and 1 Hz to stimulate outflow of tritium, a frequency-dependent suppression of responses to the second and the following pulses was observed. In the presence of the alpha-2 adrenoceptor antagonist idazoxan (10(-6) M), comparable amounts of tritium were released by the first stimulus and each of the following stimuli at 0.02 Hz. In contrast, at 0.08, 0.3 and 1 Hz the amount of 3H-overflow evoked by the first pulse was not reached in response to the following pulses. Clonidine (10(-6) M) diminished markedly the response to the first as well as to the following stimuli, irrespective of the frequency of stimulation. 3) Using two consecutive pulses delivered with decreasing pulse intervals, an apparent reduction or complete abolition of autoinhibition was observed at intervals of less than 100 msec, indicated by reduction or loss of the facilitatory effects of alpha-2 adrenoceptor antagonists. The present results provide detailed insights in operational characteristics of alpha-2 adrenoceptor-mediated autoinhibition and the effects of drugs on this regulatory mechism.

[Hemodynamics and metabolic parameters during use of the antihypertensive agent diazoxide]. / Hämodynamik und Stoffwechselparameter bei Anwendung des Antihypertonikums Diazoxid.

The antihypertensive remedy diazoxide (hyperstat) was applied in 11 patients with hypertension of the clinical stages III and IV. The patients were applied a single intravenous injection of 20 ml = 300 mg diazoxide. The behaviour of blood pressure, pulse, blood sugar and cortisol level as well as of the blood supply in rest of the musculature of the extremities was tested and evaluated. Within 1 minute after the injection the systolic blood pressure decreased by 12%, the diastolic by 20%. After an above all show increase of the diastolic blood pressure once more a slight decrease of the systolic blood pressure followed after 90 minutes, which lasted several hours. Also after 24 hours the blood pressure did not reach the original value. The increase of the pulse rate was clinically not relevant. The blood glucose increased in the 5th minute, after 15 minutes it reached its culmination point and, beginning with the 30th minute it showed a decreasing tendency. The initial values were got after 180 minutes. After the 15th minute began a relative short-term decrease of the plasma cortisol, which, however, again increased after 60 minutes. On the other hand the occlusion-plethysmographic investigations of the veins showed an increasing blood flow in rest up to 3 hours after the injection.

Estimation of agonist dissociation constants at central presynaptic alpha-2 autoreceptors in the absence of autoinhibition.

The agonist dissociation constants (KA) and relative efficacies of UK-14304, norepinephrine (NE) and clonidine at presynaptic release-modulating alpha-2 adrenoceptors were determined in rat cerebral cortex slices using the irreversible alpha-2 adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (0.8 mg/kg i.p.) to reduce the receptor pool. Eighteen hours after treatment, the slices were incubated with [3H]NE, superfused in the presence of reuptake inhibitor and stimulated electrically with short bursts of 4 pulses delivered at 100 Hz (pseudo-one-pulse; POP) or trains of 36 or 72 pulses delivered at 3 Hz. EEDQ treatment did not affect the overflow of radioactivity evoked by POP, but greatly facilitated overflow at 36 or 72 pulses/3 Hz indicating that the autoinhibition seen under the latter conditions is totally lacking with POP stimulation. KA values determined using 4 pulses/100 Hz were 136, 50 and 625 nM for UK-14304, clonidine and NE, respectively. At 36 or 72 pulses/3 Hz the values were higher by a factor of up to 3. The percentage of receptors active after EEDQ treatment was found to be 5.5 to 8.2% and was not influenced by conditions of stimulation. Receptor reserves were estimated to be about 65% for UK-14304 and NE and 40% for clonidine. The efficacies of UK-14304 and clonidine relative to NE were 1 and 0.5, respectively. The data indicate that KA values for agonists at presynaptic alpha-2 autoreceptors are inevitably underestimated if the released transmitter causes inhibition of release in addition to the drug under investigation.

Cholera toxin induces cyclic AMP-independent down-regulation of Gs alpha and sensitization of alpha 2-autoreceptors in chick sympathetic neurons.

The role of the stimulatory GTP-binding protein (Gs) in the alpha 2-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The alpha 2-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [3H] noradrenaline release with half-maximal effects at 14.0 +/- 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 +/- 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 microM forskolin or 100 microM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of alpha-subunits of Gs (Gs alpha) from particulate to soluble fractions and led ultimately to a complete loss of Gs alpha from the neurons. In contrast, no effect was seen on the distribution of either alpha-subunits of Gi- or Ga-type G proteins or of beta-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of Gs alpha. This down-regulation of Gs alpha is associated with the sensitization of alpha 2-autoreceptors.

Anger and anxiety in borderline hypertension.

Psychologic studies of hypertension have usually focused on the relationship of anger and anxiety to clinic or laboratory blood pressure (BP). Yet, average blood pressure outside of the clinic has proven to be a more important predictor of hypertensive complications. In this study, we have isolated two groups of borderline hypertensives--one group that maintained high blood pressure outside of the clinic and another whose average BP returned to normal at home. All 33 subjects were given psychometric instruments for measuring various components of anger and anxiety: Spielberger's State-Trait Personality Inventory, the Anger Expression Scale, and the State Anger Reaction Scale. The high home BP group reported greater intensity of anger, although they suppressed their expression of anger to a greater extent. The groups did not differ in anxiety. Also, blood pressure variability was not different between the two groups. It is suggested that the psychologic differences found in the group of higher-risk borderline hypertensives may, through autonomic arousal, contribute to the later development of established hypertension.