Insights into enzyme function from studies on mutants of dihydrofolate reductase.

Kinetic analysis and protein mutagenesis allow the importance of individual amino acids in ligand binding and catalysis to be assessed. A kinetic analysis has shown that the reaction catalyzed by dihydrofolate reductase is optimized with respect to product flux, which in turn is predetermined by the active-site hydrophobic surface. Protein mutagenesis has revealed that specific hydrophobic residues contribute 2 to 5 kilocalories per mole to ligand binding and catalysis. The extent to which perturbations within this active-site ensemble may affect catalysis is discussed in terms of the constraints imposed by the energy surface for the reaction.

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.

Thermoregulatory actions of centrally-administered vasopressin in the rat.

Dependent upon the route and/or site of administration, arginine vasopressin (AVP) evoked a number of thermoregulatory actions in the conscious rat. Infused into a lateral cerebral ventricle, arginine vasopressin produced short-lasting hypothermia of rapid onset. Injected into the preoptic area, arginine vasopressin caused long-lasting hyperthermia of rapid onset that was antagonized by the prior administration of a V1 receptor antagonist, [d(CH2)5 Tyr(Me)AVP]. Injections of arginine vasopressin into the nucleus accumbens, ventral septal area, substantia innominata and the dorsomedial hypothalamus were without effect on body temperature. Although the antipyretic action of arginine vasopressin within the ventral septal area has been well documented, these findings provide further evidence that this peptide exerts additional thermoregulatory actions that are both neuroanatomically and functionally specific.

Activation of the micturition reflex by NK2 receptor stimulation in the anaesthetized guinea-pig.

1. The mechanisms underlying stimulation of bladder contractions and bronchoconstriction by the selective NK2 receptor agonist, [beta-Ala8]NKA(4-10), were examined in the anaesthetized guinea-pig. 2. Atropine, alpha,beta-methylene-ATP and ganglion blocking agents were used to examine the contribution of reflex arc activation and/or potentiation of efferent mechanisms to the NK2 receptor-mediated responses seen in these two tissues. 3. [beta-Ala8]NKA(4-10)-induced bronchoconstriction was immediate, dose-dependent and was unaffected by pretreatment with ganglion blockers (hexamethonium or chlorisondamine), blockade of muscarinic receptors by atropine, or desensitization of P2 purinoceptors by alpha,beta-methylene-ATP. 4. At does of 5 micrograms kg-1 and above, [beta-Ala8]NKA(4-10) induced bladder contractions that appeared to be of an 'all-or-nothing' nature. These contractions occurred after a delay of 10 to 30 s and were often biphasic, comprised of an initial rapid component followed by a slower tonic component. 5. Pretreatment of the animals with either atropine or the desensitizing purinoceptor agonist alpha,beta-methylene-ATP, resulted in partial inhibition of bladder contractile responses to [beta-Ala8]NKA(4-10). The combination of atropine and alpha,beta-methylene-ATP pretreatment resulted in additive inhibition leading to complete blockade of the response. 6. The bladder responses to [beta-Ala8]NKA(4-10) (5 micrograms kg-1) were inhibited by pretreatment with the ganglion blockers, hexamethonium and chlorisondamine, indicating a preganglionic mechanism of action. 7. These findings demonstrate the indirect nature of the bladder contractions induced by activation of NK2 receptors in the anaesthetized guinea-pig. Contractions occur secondary to the release of endogenous cholinergic and NANC transmitters by activation of neuronal NK2 receptors located at apreganglionic site, possibly on capsaicin-sensitive sensory afferent nerves, where NK2 sites have been demonstrated autoradiographically. In contrast, [beta-Ala8]NKA(4- 10)-induced bronchoconstriction in the anaesthetized guinea-pig is a direct smooth muscle contractile response that is unaffected by ganglionblockade or blockade of muscarinic receptors.

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes. 6. Noradrenaline-mediated contractions of rat aorta were sensitive to the alkylating effects of chlorethylclonidine (CEC). CEC (10 microM) caused a small rightward shift in the noradrenaline concentration-response curve. CEC at 100 microM caused a further shift and suppression of the maximum response to noradrenaline.7. The results of this study suggest that noradrenaline predominantly, but not exclusively, mediates contraction of rat aorta through the activation of an alphalD-adrenoceptor.

Pharmacological properties of the cloned alpha 1A/D-adrenoceptor subtype are consistent with the alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

1. The pharmacological characteristics of cloned mammalian alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptor subtypes expressed in rat 1 fibroblasts were determined in comparison to the binding and functional properties of these subtypes in rat tissues. 2. Analysis of [3H]-prazosin binding to membrane homogenates from rat 1 fibroblast cells expressing each of the alpha 1-subtypes indicated high affinity binding to a single population of binding sites. Binding affinities were similar for alpha 1A/D-, alpha 1B- and alpha 1C-subtypes (Kds: 0.13, 0.10 and 0.15 nM respectively) although a higher density of alpha 1B- and alpha 1C-receptors (Bmax: 4068 and 10,323 fmol mg-1 protein respectively) were expressed in comparison to alpha 1A/D (838 fmol mg-1). 3. Displacement of [3H]-prazosin from membranes expressing cloned alpha 1-adrenoceptor subtypes revealed that 5-methyl-urapidil, WB 4101, benoxathian and phentolamine displayed high affinity and selectivity for alpha 1A/D- over alpha 1B-subtypes. These compounds also had high affinity and selectivity for alpha 1C- over alpha 1B-subtypes. 5-Methyl-urapidil showed selectivity for alpha 1C (Ki 0.60 +/- 0.16 nM) over both alpha 1A/D (Ki, 9.8 +/- 2.8 nM) and alpha 1B (Ki 57.2 +/- 12 nM) subtypes. Prazosin and doxazosin were not subtype selective. 4. In comparison to [3H]-prazosin a similar pharmacological profile was obtained with [125I]-HEAT using cloned alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptors expressed in rat 1 fibroblasts. 5. The affinities of prazosin, WB 4101, 5-methyl-urapidil, phentolamine and benoxathian at cloned alpha 1A/D-receptors were consistent with alpha 1A affinities determined with chlorethylclonidine-treated rat cortical membranes. Affinities at cloned XIB-receptors were consistent with alpha 1B affinities determined with rat liver membranes.6. Using the epididymal rat vas deferens as a functional measure of alpha 1A affinity, prazosin (pA29.23 +/- 0.28), WB 4101 (pA2 9.58 +/- 0.12), phentolamine (pKB 7.90 +/- 0.16), benoxathian (pKB 9.21 +/- 0.21)and 5-methyl-urapadil (pKB 8.51 +/-0.16) were potent antagonists of noradrenaline-induced contractions.7. At present, evidence from cloning studies suggests the existence of at least three alpha 1-adrenoceptor subtypes. In contrast to the recent proposal for alpha l-adrenoceptor classification, the pharmacology of the cloned alpha 1A/D (or alpha lD)-adrenoceptor is more consistent with that of an alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

Evaluation of the pharmacological selectivity profile of alpha 1 adrenoceptor antagonists at prostatic alpha 1 adrenoceptors: binding, functional and in vivo studies.

1. The profile of a range of alpha 1 adrenoceptor antagonists was determined in vitro against cloned human alpha 1A, alpha 1B and alpha 1D adrenoceptors and against noradrenaline-mediated contractions of rat aorta and human prostate. The in vivo profile of compounds was determined in an anaesthetized dog model which allowed the simultaneous assessment of antagonist potency against phenylephrine-mediated increases in blood pressure and prostatic pressure. 2. The quinazoline antagonists, prazosin, doxazosin and alfuzosin displayed high affinity but were non selective for the three cloned human alpha 1 adrenoceptors. Indoramin and SNAP 1069 showed selectivity for alpha 1A and alpha 1B adrenoceptors relative to the alpha 1D subtype. Rec 15/2739, WB 4101, SL 89,0591, (+)- and (-)- tamsulosin showed selectivity for alpha 1A and alpha 1D adrenoceptors relative to the alpha 1B subtype. RS 17053 showed high affinity and selectivity for alpha 1A adrenoceptors (pKi 8.6) relative to alpha 1B (pKi = 7.3) and alpha 1D (pKi = 7.1) subtypes. 3. (+)-Tamsulosin, (-)-tamsulosin, SL 89,0591, Rec 15/2739, SNAP 1069 and RS 17053 appeared to act as competitive antagonists of noradrenaline-mediated contractions of rat aorta yielding pA2 affinity estimates which were similar to binding affinities at cloned human alpha 1D adrenoceptors. The following rank order was obtained: prazosin = (-)-tamsulosin > doxazosin > SL 89,0591 = (+)-tamsulosin > Rec 15/2739 > RS 17053 = SNAP 1069. 4. (-)-Tamsulosin was a very potent, insurmountable antagonist of noradrenaline-mediated contractions of human prostate, yielding an approximate pA2 estimate of 9.8 at 1 nM. The corresponding (+)-enantiomer was 30 fold weaker. SL 89,0591, SNAP 1069 and Rec 15/2739 yielded pA2 estimates which compared well with their alpha 1A binding affinities. The affinity estimate for prazosin on human prostate was lower than the corresponding binding affinity determined at alpha 1A adrenoceptors and RS 17053 was a very weak antagonist on human prostate (pA2 = 6.0) relative to the high affinity (pKi = 8.6) determined at cloned human alpha 1A adrenoceptors. 5. In the anaesthetized dog, in vivo pseudo "pA2' values showed that doxazosin, (+)- and (-)-tamsulosin inhibited phenylephrine-induced increases in prostatic and blood pressure with similar affinity, implying that these agents show little or no selectivity for prostatic responses in this model. SL 89,0591 and SNAP 1069 were moderately selective (3 and 6 fold respectively) for prostatic pressure relative to blood pressure. Rec 15/2739 was a more potent antagonist of phenylephrine-mediated increases in prostatic pressure ("pA2' = 8.74) compared to blood pressure ("pA2' = 7.51). 6. Data in this study suggest that the alpha 1 adrenoceptor mediating noradrenaline-induced contractions of human prostate, whilst having some of the characteristics of an alpha 1A adrenoceptor, cannot be satisfactorily aligned with cloned alpha 1A, alpha 1B or alpha 1D adrenoceptors. In addition, studies in the anaesthetized dog have shown that agents having high affinity and selectivity for prostatic alpha 1 adrenoceptors, particularly over the alpha 1D subtype, appear to inhibit phenylephrine-induced increases in prostatic pressure selectively compared to blood pressure.

Central administration of corticotrophin-releasing factor in the sheep: effects on secretion of gonadotrophins, prolactin and cortisol.

Stress interferes with the normal secretion of LH and FSH from the anterior pituitary gland and therefore exerts a deleterious effect on reproductive function. Evidence suggests that the stress-induced disruption of gonadal function is due to a central action of corticotrophin-releasing factor (CRF) to inhibit the release of LHRH into the hypophysial-portal circulation. The following studies were undertaken to investigate further the role of CRF in regulating gonadotrophin release in the sheep and to determine whether central administration of this peptide can alter the secretion of other hormones (e.g. prolactin and cortisol) known to be released under conditions of stress. In contrast to other species, injection of CRF into the third ventricle of the sheep brain caused a dose-related stimulation of LH secretion. The pulse frequency and mean levels of LH were increased significantly following central administration of CRF. In contrast to this effect, central administration of CRF did not alter the plasma concentration of FSH but caused a marked and dose-related stimulation of prolactin and cortisol secretion. The stimulatory effect of CRF on prolactin secretion was reversed by i.v. administration of the opioid antagonist naloxone, suggesting that endogenous opioid peptides mediate the central effect of CRF on the release of prolactin, but not cortisol. In conclusion, these data demonstrate that administration of CRF causes a dose-related stimulation of LH and prolactin release from the anterior pituitary gland and cortisol from the adrenal gland. In the case of prolactin, endogenous opioid peptides are likely to mediate this response.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of pyrogen fevers are altered in the aged rabbit.

The febrile response to both intravenous and intracerebral administration of pyrogens was investigated in young and old male New Zealand White rabbits. Intravenous bacterial pyrogen evoked biphasic fevers in both groups of animals. However, the fevers in the group of older rabbits were significantly less than in younger animals. In contrast, intravenous injection of endogenous pyrogen produced identical fevers in the two groups. Bacterial and endogenous pyrogens injected into a lateral cerebral ventricle evoked marked febrile responses of long duration in both young and old rabbits. The responses of the old rabbits were significantly less than those of the younger ones. Finally, direct microinjection of prostaglandin E1 into tissue sites within the anterior hypothalamic preoptic area elicited short latency hyperthermic responses which were significantly less in the older rabbits. Analysis of ear skin temperatures during fever demonstrated that some of the differences may, in part, be due to altered vasoconstrictor responses in the peripheral vasculature. Thus, these data indicate that the febrile response is altered with increasing age in the rabbit.

Evaluation of a possible role for the dopamine d and d receptors in the steroid-dependent suppression of luteinizing hormone secretion in the seasonally anoestrous ewe.

Abstract This study was undertaken to determine whether dopaminergic suppression of pulsatile luteinizing hormone (LH) secretion during seasonal anoestrus in the ewe is mediated via the dopamine D(1) or D(2) receptor. This was tested by 1) assessing the response to dopamine D(1) and D(2) antagonists during seasonal anoestrus, and 2) determining the ability of D(1) and D(2) agonists to suppress pulsatile LH secretion during the breeding season. In seasonally anoestrous ewes the D(2) antagonist pimozide increased LH pulse frequency although this effect did not reach significance (P = 0.07). The D(1) antagonist SCH 23390 had no effect on LH pulse frequency. LH pulse amplitude and mean LH were not affected by either treatment. During the breeding season, ovariectomized oestradiol-implanted ewes were injected intracerebroventricularly with vehicle, LY 171555 (dopamine D(2) agonist) and SKF 38393 (D(1) agonist) with each drug tested at 50 mug and 200 mug. At the higher dose, LY 171555 significantly (P<0.05) reduced LH pulse frequency in the 2 h period immediately after treatment. Mean LH declined at both doses but only in the first hour after treatment. SKF 38393 did not affect LH pulse frequency, pulse amplitude or mean LH. These results suggest that the D(1) receptor is not involved in the suppression of pulsatile LH secretion during seasonal anoestrus. Dopaminergic suppression of pulsatile LH secretion is mediated via the D(2) receptor but the significance of this neurotransmitter in the seasonal suppression of LH remains to be elucidated.

N-Methyl-DL-Aspartate Receptor Antagonism by D-2-Amino-5-Phosphonovaleric Acid Delays Onset of Puberty in the Female Rat.

Abstract To investigate the possible role of N-methyl-DL-aspartate (NMDA) receptor activation in the initiation of puberty, we examined the effects of the selective competitive antagonist 2-amino-5-phosphonovaleric acid (AP5) on the timing of vaginal opening. Paired and weight-matched litter mates of immature female rats were implanted with osmotic minipumps for the intracerebroventricular infusion of DL- or D-AP5 or artificial cerebrospinal fluid from 27 to 30 days of age for 14 days. Each animal was weighed and examined daily for vaginal opening as the indicator of first oestrous. Infusion of 20 or 40 mM DL-AP5 beginning on Day 30 failed to delay vaginal opening. Administration 50mM of the single enantiomer D-AP5 beginning on Day 27 significantly delayed the age of vaginal opening to 40.6+/-1.1 (mean +/- SEM) days compared to the cerebrospinal fluid-infused controls (36.5 +/- 0.6 days). Blockade of NMDA receptors in the D-AP5-treated animals was confirmed on Day 32 by the suppression of luteinizing hormone response to intravenous NMDA (20 mg/kg) while the response to exogenous luteinizing hormone-releasing hormone (50 ng/kg) remained intact. AP5-treated animals had a slower rate of growth (3.1 +/- 0.2 g/day) compared to controls (4.2 +/- 0.2 g/day). However, a similar degree of growth retardation produced by a 75% restricted diet in untreated juvenile animals did not delay vaginal opening. This suggests that the slower growth rate in the D-AP5-treated animals could not account for the delayed onset of puberty. In conclusion, these data suggest that blockade of central NMDA receptors inhibits excitatory mechanisms which may be important in the control of pubertal onset in the female rat.

Naloxone does not Affect the Luteinizing Hormone-Releasing Hormone-Induced Inhibition of Luteinizing Hormone Secretion in Sheep.

Abstract Injection of luteinizing hormone-releasing hormone (21 pmol) into the third cerebral ventricle of long-term ovariectomized ewes caused a marked inhibition of luteinizing hormone secretion. Mean luteinizing hormone levels and luteinizing hormone pulse frequency were reduced significantly when compared with the control responses to saline (50 mul). A notable characteristic of the response was the delayed and sustained nature of the luteinizing hormone-releasing hormone-induced inhibition. In the presence of the opioid antagonist naloxone (4 +/- 25 mg iv), the central administration of luteinizing hormone-releasing hormone still produced a marked inhibition of luteinizing hormone secretion. Again, mean luteinizing hormone levels and luteinizing hormone pulse frequency were reduced significantly. When naloxone was injected iv, there was a significant rise in mean luteinizing hormone levels as a consequence of an increase in pulse frequency (in four out of five ewes) and a significant increase in luteinizing hormone pulse amplitude. In conclusion, these data suggest that central opioid pathways sensitive to blockade by naloxone are not involved in the luteinizing hormone-releasing hormone-induced inhibition of luteinizing hormone release. Furthermore, in the long-term ovariectomized ewe, endogenous opioid peptides exert a tonic inhibitory influence on luteinizing hormone-releasing hormone/luteinizing hormone secretion.

Effects of Beta-endorphin on pulsatile luteinizing hormone and prolactin secretion during the follicular phase in the ewe.

Abstract The present study was undertaken to determine the effects of the endogenous opioid ligandbeta-endorphin on pulsatile luteinizing hormone (LH) secretion and plasma prolactin concentrations during the follicular phase of the ewe. Oestrous cycles were synchronized by injection of prostaglandin analogue and, commencing 13 h later, saline or beta-endorphin (2, 10 or 50 mug) was injected intracerebroventricularly at hourly intervals for 3 h. Treatment with beta-endorphin was followed by a significant reduction in LH pulse frequency at all doses due to almost complete cessation of pulses. There were no significant changes in LH pulse amplitude or mean LH concentrations. At the lowest dose ofbeta-endorphin, LH pulses recommenced within 3 h of the last injection in all animals and pulse frequency was not significantly different from the saline-injected controls during the 3 h post-treatment period. Following treatment with 10 or 50 mug beta-endorphin, LH pulse frequency remained suppressed during the 3 h post-treatment period but was not different from saline-treated controls on the following day. The time to the onset of the LH surge was not affected by intracerebroventricularbeta-endorphin. Plasma prolactin concentrations were significantly increased following intracereb-roventricular injection of 10 or 50 mug beta-endorphin, declining to control values soon after treatments stopped. Intravenous administration of 50 mug beta-endorphin had no effect on LH but was accompanied by a small increase in prolactin concentrations. While these results indicate that hypothalamicbeta-endorphin may be involved in the central control of LH and prolactin secretion, they provide no evidence for subtle modulation of LH pulse frequency by this neuropeptide.

Endocrine actions of central neuropeptide Y in the ewe: activation of the hypothalamo-pituitary-adrenal axis by exogenous neuropeptide Y and role of endogenous neuropeptide Y in the secretion of luteinizing hormone during the oestrous cycle.

Neurons immunoreactive for neuropeptide Y (NPY) are abundant in the hypophysiotrophic areas of the brain. In particular, there is considerable anatomical evidence for the influence of this neuropeptide on the reproductive and hypothalamo-pituitary-adrenal axes. We therefore investigated whether central administration of NPY can alter the activities of the reproductive and hypothalamopituitary-adrenal axes in the ewe, and whether ovarian steroids are involved in the modulation of these events. We also attempted to investigate whether endogenous NPY is important in the control of luteinizing hormone-releasing hormone/luteinizing hormone (LHRH/LH) secretion in the sheep oestrous cycle. Central injection of NPY (0.15 and 1.5 nmol in 50 microliters saline), delivered by gravity flow into the third cerebral ventricle, had no effect on LH levels in ovariectomized (OVX) ewes (n = 6) or OVX ewes implanted with oestradiol (OVX/E2) (n = 7), nor was LH secretion altered by central NPY (1.5 nmol) in intact cycling animals in either the follicular or the luteal phase (n = 5). However, central administration of 1.5 nmol NPY to intact ewes during both the follicular (P < 0.05) and the luteal phase (P < 0.01), and in OVX/E2 ewes (P < 0.05) caused a large and significant increase in plasma cortisol levels. High titre antibodies were raised to NPY in sheep and the effects of peripheral and central (intracerebroventricular) administration of anti-NPY antibodies on the timing and/or characteristics of the E2-induced LH surge in anoestrous ewes and of the preovulatory surge of LH in cycling ewes were determined. Intravenous administration of anti-NPY antibodies (n = 6) had no effect on the oestradiol benzoate-induced LH surge, compared with the control injection of non-immune plasma (n = 6). Likewise, passive systemic immunization against NPY (n = 10) was without effect on the characteristics of the preovulatory LH surge, compared with the control group (n = 10). However, central (intracerebroventricular) administration of anti-NPY antibodies (n = 4) delayed or abolished the preovulatory LH surge when compared with non-immune plasma treatment in the same animals. In summary, tonic LHRH/LH secretion is unaffected by centrally administered NPY at the doses used in this study. However, the same doses of NPY activate the hypothalamo-pituitary-adrenal axis, thus lending clear support to the hypothesis that NPY is involved in the multifactorial regulation of adrenocorticotrophin and cortisol secretion in this species, probably by stimulating corticotrophin-releasing hormone and/or arginine vasopressin secretion within the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide-Y stimulates pituitary-adrenal activity in fetal and adult sheep.

Corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) are the primary neuropeptides regulating the secretion of ACTH from the anterior pituitary gland during fetal and adult life. However, a number of other neuropeptides including neuropeptide-Y (NPY) appear to modulate the activity of this system. The potential role of NPY in the regulation of pituitary-adrenal function was examined in fetal and adult sheep. Administration of NPY (6.5 micrograms) as a bolus injection into the third cerebral ventricle of adult ewes elicited a significant (P < 0.05) increase in plasma concentrations of ACTH. In fetal sheep at day 125 gestation (term = 145 days) a five-fold higher dose (30 micrograms) of NPY injected into the lateral cerebral ventricles also caused a significant increase in plasma concentrations of ACTH. The potential of NPY to influence ACTH secretion directly from the pituitary gland was investigated using primary cultures of fetal (day 130 gestation) and adult pituitary cells. CRF (10(-10)-10(-7) M) caused a significant (P < 0.01) dose-related increase in ACTH secretion from both fetal and adult pituitary cells. Furthermore, the secretion of ACTH from adult cells was significantly greater (P < 0.05) than that from fetal cells. NPY (10(-10)-10(-7) M) had no effect on basal or CRF-stimulated ACTH secretion from fetal or adult pituitary cells. Pre-incubation of pituitary cells with cortisol (10(-9) and 10(-7) M) for three days significantly inhibited CRF-stimulated ACTH secretion but had no effect on basal ACTH release. The simultaneous addition of NPY did not alter the ability of cortisol to inhibit CRF-stimulated ACTH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between neuropeptide Y, luteinizing hormone-releasing hormone and estradiol in the control of luteinizing hormone release from cultured ovine pituitary cells.

This study used pituitary cells in culture firstly to test the hypothesis that NPY may augment the pituitary LH response to LHRH and secondly to determine whether this interaction is dependent on the presence of estradiol. LHRH (10(-10)-10(-6) M) caused a significant increase in LH secretion from dispersed ovine pituitary cells maintained in culture for six days, a response which was enhanced when cells were pretreated for three days with 4 x 10(-11) M estradiol. NPY 10(-10)-10(-6) M) had no effect on basal LH release from ovine pituitary cells maintained either in the presence or absence of estradiol. NPY (10(-10) and 10(-8) M) also had no effect on LHRH-stimulated LH release either in the presence or absence of estradiol. These results substantiate previous observations that physiologically relevant concentrations of estradiol enhance the LH response to LHRH in cultured ovine pituitary cells. However, in contrast to experiments carried out using rat pituitary cells in culture, the present data provide no evidence to support the hypothesis that NPY alone interacts with LHRH in the control of LH secretion from the ovine pituitary gland.

Effect of alpha 1 adrenoceptor antagonists on prostatic pressure and blood pressure in the anesthetized dog.

OBJECTIVES: In the current study we have profiled a range of compounds at alpha 1 adrenoceptor subtypes in vitro and have assessed their effects in vivo using the anesthetized dog in an attempt to elucidate the predominant alpha 1 adrenoceptor subtype mediating contractile responses of the canine prostate. METHODS: The affinity of compounds for alpha 1 adrenoceptor subtypes was determined by displacement of [3H] prazosin binding from stably transfected rat 1 fibroblasts expressing alpha 1A, alpha 1B, and alpha 1C, adrenoceptor subtypes. The potency of these agents was then assessed in vivo using an anesthetized dog model allowing simultaneous measurement of prostatic pressure and blood pressure following intravenous (i.v.) administration of phenylephrine (1 to 128 micrograms/kg). RESULTS: All compounds examined in this study showed high and similar affinity for alpha 1 adrenoceptor subtypes, with the exception of 5-Methyl-urapidil, which was selective for alpha 1C (pKi = 9.3) over alpha 1B (pKi = 7.2) and alpha 1A (pKi = 8.1). Doxazosin, terazosin, alfuzosin, and tamsulosin were potent antagonists of phenylephrine responses and in vivo derived "pseudo pA2" determinations showed that the drugs did not discriminate between prostatic and vascular receptors. 5-Methyl-urapidil was also a potent antagonist of phenylephrine-induced responses but was selective for prostatic pressure ("pseudo pA2" = 8.7) over blood pressure ("pseudo pA2" = 7.2). CONCLUSIONS: Data in the present study suggest a predominant role of the alpha 1C adrenoceptor subtype in the contractile response of the canine prostate to phenylephrine in vivo. This model therefore provides a suitable means of assessing putative prostate-selective antagonists for the treatment of benign prostatic hyperplasia.

Antipyretic action of centrally administered arginine vasopressin but not oxytocin in the cat.

The antipyretic action of central arginine vasopressin (AVP) was investigated in mongrel cats. Control push-pull perfusions in the ventral septal area (VSA), with the carrier vehicle alone, did not affect the febrile response to Salmonella typhosa administered intracerebroventricularly. When AVP was perfused similarly, the fever was suppressed in a dose-related manner. The lower dose of AVP delayed the onset of fever, whereas the higher concentration of AVP suppressed consistently the fever throughout the period of administration. Another neurohypophyseal peptide, oxytocin, was ineffective in altering the febrile response at the dose tested. The regions of greatest sensitivity to the antipyretic action of AVP are located ventral to the septum, bounded by the diagonal bands of Broca, extending into the posterior septal nucleus. Sites at which AVP was ineffective in producing antipyresis were found more dorsal and lateral to these. Thus, AVP suppresses fever in the cat via an action in the VSA that is dose related, and site specific and peptide specific. These data provide further evidence that AVP may be involved in the central mechanisms which control core temperature.

Perfusion of vasopressin within the rat brain suppresses prostaglandin E-hyperthermia.

These experiments were undertaken to determine whether arginine vasopressin (AVP) could suppress a prostaglandin hyperthermia and to localize sites of these actions in the rat. Prostaglandin E2 (PGE2) sensitive sites were localized in the ventral-septal area by microinjecting 200 ng/0.5 microliter of prostaglandin E2. During perfusion with an artificial CSF, PGE2 injected into the lateral cerebral ventricle evoked a hyperthermia of more than 1 degree C. Perfusion of 6.5 micrograms/ml of AVP markedly attenuated the PGE2-induced hyperthermia. These results suggest that AVP suppresses PGE2-induced hyperthermia in sites in which PGE2 evokes an increase in core temperature.

Central effects of vasopressin and 1-desamino-8-D-arginine vasopressin (DDAVP) on interleukin-1 fever in the rat.

The intracerebroventricular administration of arginine vasopressin suppressed significantly the fever evoked by interleukin-1. This antipyretic action of arginine vasopressin was blocked completely by the antivasopressor analog d(CH2)5Tyr(Me)arginine vasopressin, an antagonist of the V1 subtype of peripheral vasopressin receptor. However, in contrast to AVP, the V2 receptor agonist, 1-desamino-8-D-arginine vasopressin, did not alter the normal time course or magnitude of interleukin-1 fever. These data suggest that arginine vasopressin induced antipyresis is mediated via central receptors which may resemble the V1 subtype of peripheral vasopressin receptor. The V2 subtype of vasopressin receptor is unlikely to be involved since an agonist of this receptor did not exhibit any antipyretic activity against interleukin-1 fever.