Melanocortins and cardiovascular regulation.

The melanocortins form a family of pro-opiomelanocortin-derived peptides that have the melanocyte-stimulating hormone (MSH) core sequence, His-Phe-Arg-Trp, in common. Melanocortins have been described as having a variety of cardiovascular effects. We review here what is known about the sites and mechanisms of action of the melanocortins with respect to their effects on cardiovascular function, with special attention to the effects of the gamma-melanocyte-stimulating hormones (gamma-MSHs). This is done in the context of present knowledge about agonist selectivity and localisation of the five melanocortin receptor subtypes cloned so far. gamma2-MSH, its des-Gly12 analog (= gamma1-MSH) and Lys-gamma2-MSH are 5-10 times more potent than adrenocorticotropic hormone-(4-10)(ACTH-(4-10)) to induce a pressor and tachycardiac effect following intravenous administration. The Arg-Phe sequence near the C-terminal seems to be important for full in vivo intrinsic activity. Related peptides with a C-terminal extension with (gamma3-MSH) or without the Arg-Phe sequence (alpha-MSH, as well as the potent alpha-MSH analog, [Nle4,D-Phe7]alpha-MSH), are, however, devoid of these effects. In contrast, ACTH-(1-24) has a depressor effect combined with a tachycardiac effect, effects which are not dependent on the presence of the adrenals. Although the melanocortin MC3 receptor is the only melanocortin receptor subtype for which gamma2-MSH is selective, in vivo and in vitro structure-activity data indicate that it is not via this receptor that this peptide and related peptides exert either their pressor and tachycardiac effects or their extra- and intracranial blood flow increasing effect. We review evidence that the pressor and tachycardiac effects of the gamma-MSHs are due to an increase of sympathetic outflow to the vasculature and the heart, secondary to activation of centrally located receptors. These receptors are most likely localised in the anteroventral third ventricle (AV3V) region, a brain region situated outside the blood-brain barrier, and to which circulating peptides have access. These receptors might be melanocortin receptors of a subtype yet to be identified. Alternatively, they might be related to other receptors for which peptides with a C-terminal Arg-Phe sequence have affinity, such as the neuropeptide FF receptor and the recently discovered FMRFamide receptor. Melanocortin MC4 receptors and still unidentified receptors are part of the circuitry in the medulla oblongata which is involved in the depressor and bradycardiac effect of the melanocortins, probably via interference with autonomic outflow. Regarding the effects of the gamma-MSHs on cortical cerebral blood flow, it is not yet clear whether they involve activation of the sympathetic nervous system or activation of melanocortin receptors located on the cerebral vasculature. The depressor effect observed following intravenous administration of ACTH-(1-24) is thought to be due to activation of melanocortin MC2 receptors whose location may be within the peripheral vasculature. Melanocortins have been observed to improve cardiovascular function and survival time in experimental hemorrhagic shock in various species. Though ACTH-(1-24) is the most potent melanocortin in this model, alpha-MSH and [Nle4,D-Phe7]alpha-MSH and ACTH-(4-10) are quite effective as well. As ACTH-(4-10) is a rather weak agonist of all melanocortin receptors, it is difficult to determine via which of the melanocortin receptors the melanocortins bring about this effect. Research into the nature of the receptors involved in the various cardiovascular effects of the melanocortins would greatly benefit from the availability of selective melanocortin receptor antagonists.

A study on possible modulating and direct effects of gamma2-MSH and ACTH-(1-24) on the cardiovascular system of the rat.

In conscious rats, gamma2-melanocyte-stimulating hormone (gamma2-MSH) dose-dependently increases blood pressure and heart rate, whereas adrenocorticotropin-(1-24) [ACTH-(1-24)] dose-dependently decreases blood pressure, an effect which was accompanied by a reflectory tachycardia. As the exact mechanism involved in these cardiovascular effects of the two melanocortins is as yet not known, we undertook a series of experiments to investigate the possibility that these peptides have modulating or direct effect on the cardiovascular system of the rat. In pithed rats gamma2-MSH, administered intravenously (i.v.) in doses of 5-200 nmol/kg, had no significant effect on systolic and diastolic blood pressure and on heart rate, whereas ACTH-(1-24), 5-500 nmol/kg, i.v., dose-dependently decreased blood pressure and increased heart rate. Infusion of gamma2-MSH, 10(-8) M, or ACTH-(1-24), 10(-6) M, in the isolated perfused rat heart did not significantly affect left ventricular pressure or coronary flow. Pretreatment with either gamma2-MSH or ACTH-(1-24) did not modify the responsiveness of the myocardium and coronary vasculature to salbutamol and phenylephrine. Neither gamma2-MSH nor ACTH-(1-24) did affect the vascular contractile machinery of skinned vascular smooth muscles of the rabbit with respect to Ca2+ handling in the cell, as measured by its sensitivity to exogenously applied Ca2+. Gamma2-MSH had no effect on blood pressure and heart rate in pithed rats in which postganglionic sympathetic outflow was stimulated by 1,1-dimethyl-4-phenylpiperazinium (DMPP), nor in pithed rats in which preganglionic sympathetic outflow was stimulated electrically. A dose of 15 nmol/kg ACTH-(1-24) had no significant influence on preganglionic outflow to the cardiac and vascular structures in pithed rats. These data show that gamma2-MSH does not exert its cardiovascular effects via a peripheral site of action at the level of the vascular system and the heart, nor directly on pre- or postganglionic sympathetic outflow. These results are in support for the notion that the peptide acts via a brain region localised outside the blood-brain barrier. The acute depressor effect of ACTH-(1-24), however, seems to be due to a direct effect on the vasculature in the periphery.

The effect of the adrenocorticotropin-(4-9) analogue, ORG 2766, and of dizolcipine (MK-801) on infarct volume in rat brain.

The purpose of this study was to evaluate whether the synthetic adrenocorticotropin-(4-9) (ACTH-(4-9)) analogue ORG 2766, HMet(O2)-Glu-His-Phe-D-Lys-Phe-OH, which has been shown to have beneficial effects on both the recovery from experimentally induced lesions of the central nervous system and peripheral nerve degeneration, has a protective effect on focal ischemic neuronal damage. The NMDA receptor antagonist dizolcipine (MK-801), a very potent neuroprotective drug, was used as positive reference compound. Isoflurane-anesthetized rats had the middle cerebral artery occluded using either an intravasal or an extravasal technique, because pilot experiments had shown differences in the severity of ischemia for the two middle cerebral artery occlusion techniques. MK-801, 500 microg kg(-1) min(-1), or saline was administered i.v. 30 min after occlusion of the middle cerebral artery. In the ACTH-(4-9) analogue/saline group, 10 and 150 microg/kg of the analogue, or saline was injected s.c. both directly after and 24 h after occlusion. The ACTH-(4-9) analogue treatment had no effect on the infarction volume in either model of middle cerebral artery occlusion, whereas MK-801 caused a significant reduction in the volume of cortical infarction in both models. We conclude that, although ORG 2766 is known to enhance the recovery from experimentally induced lesions of the central nervous system through a neurotrophic action and has proven to have significant beneficial effects on peripheral nerve regeneration, it did not prevent ischemic neuronal damage after intravasal or extravasal middle cerebral artery occlusion in rats. The results with MK-801, which caused significant reductions in the volume of cortical infarction in both models of middle cerebral artery occlusion, with clearly the largest reduction in the intravasal middle cerebral artery occlusion model, again indicate that there are differences in the severity of the cerebral ischemia which the two models produce in the rat brain.

Collateral hemodynamics after middle cerebral artery occlusion in Wistar and Fischer-344 rats.

We investigated whether the difference in infarction volume after occlusion of a long proximal segment of the middle cerebral artery between Wistar and Fischer-344 rats, is caused by differences in collateral blood flow rate through leptomeningeal anastomoses. In view of the retrograde direction of collateral blood flow into the middle cerebral artery territory, we developed parasagittal laser-Doppler flowmetry. Using this method two laser-Doppler probes are placed on the cerebral cortex: probe 1 is placed near the anastomoses between the middle- and anterior cerebral artery, probe 2 is placed 2 mm further away from these anastomoses than probe 1. We found in both rat strains a comparable relation between the areas under the curve of the signal measured by both laser-Doppler probes for 2 h after middle cerebral artery occlusion. This relation is considered to be a measurement of the collateral blood flow rate into the middle cerebral artery territory through leptomeningeal anastomoses after middle cerebral artery occlusion. We conclude that collateral blood flow for the two strains were essentially similar for the initial 2 h after MCA occlusion. Although these collateral blood flows could have been different at a later time, it is unlikely that the interstrain difference in cerebral infarction volume between Wistar and Fischer-344 rats after proximal middle cerebral artery occlusion is caused by an apparent interstrain difference in the magnitude of collateral blood flow rate through leptomeningeal anastomoses. The parasagittal laser-Doppler flowmetry technique we developed for these experiments is currently successfully used in our laboratory to evaluate the efficacy of hemodynamically active pharmacotherapeutical agents in raising the collateral blood flow rate into the middle cerebral artery territory after middle cerebral artery occlusion.

Rat middle cerebral artery occlusion by an intraluminal thread compromises collateral blood flow.

We compared in Wistar rats collateral blood flow through leptomeningeal anastomoses after middle cerebral artery occlusion using craniotomy ('extravasal occlusion'), which results in a small volume of cerebral infarction, and after intraluminal thread occlusion ('intravasal occlusion'), which produces a large volume of cerebral infarction. Simultaneous laser-Doppler flowmetry with two probes placed on the cerebral cortex was used to measure and compare collateral blood flow. Extravasal occlusion caused a cortical blood flow reduction along a gradient in lateral direction, whereas blood flow reduction after intravasal occlusion was more uniformly distributed. It is concluded that permanent intravasal occlusion compromises collateral blood flow and therefore may not be a suitable model for measuring the ability of pharmacotherapeutic agents, if any, to improve collateral blood flow acutely after middle cerebral artery occlusion. The two models can be useful for testing drugs on parenchymal neuroprotective properties. Thereby, the intraluminal technique is preferred because of the possibility to study reperfusion damage when transient occlusion is applied.

Influence of blockade of alpha 1-adrenoceptors, beta 1-adrenoceptors and vasopressin V1A receptors on the cardiovascular effects of gamma 2-melanocyte-stimulating hormone (gamma 2-MSH).

gamma 2-Melanocyte-stimulating hormone (gamma 2-MSH) and related melanotropins have been shown to have various cardiovascular effects, including acute, short-lasting increases in blood pressure (MAP) and heart rate (HR). gamma 2-MSH, administered intravenously, dose-dependently increased MAP and HR with an ED50 of approximately 30 nmol/kg and a maximal effect on MAP of approximately 55 mm Hg and on HR of around 70 beats per minute. Intravenous (i.v.) pretreatment with the alpha 1-adrenoceptor antagonist, prazosin, caused the dose-response curve for the effect of gamma 2-MSH on MAP to shift to the right with a decrease in slope, whereas it had no effect on the dose-response curve for the effect on HR. I.v. pretreatment with the beta 1-adrenoceptor antagonist, metoprolol, had no effect on the dose-response curve for the effect of gamma 2-MSH on MAP, but it caused the dose-response curve for the effect of the peptide on HR to shift to the right with a decrease in slope. Neither i.v. nor intracerebroventricular (i.c.v.) administration of the vasopressin V1A receptor antagonist, SR 49059 ((2S) 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxy-benzene-sulfonyl)- 3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide), had significant effects on the dose-response curves for the effects of the peptide on either MAP or HR. The doses of prazosin, metoprolol and SR 49059 were found to be effective in counteracting the effects of agonists for these receptors (phenylephrine, isoprenaline and [Arg8]vasopressin, respectively). Taken together, these results support the postulate that the effects of gamma 2-MSH are, at least partially, due to an increase in sympathetic outflow to the periphery (Gruber and Callahan (1989), Am J Physiol 257: R681-R694), and that this increase leads to increased activation of vascular alpha 1-adrenoceptors and cardiac beta 1-adrenoceptors. If, as was suggested by these authors, gamma 2-MSH acts via activation of a central vasopressin system, it is via a vasopressin receptor subtype other than the vasopressin V1A receptor, since i.c.v. administration of a selective vasopressin V1A receptor antagonist failed to interfere with the pressor and cardioaccelerator effects of gamma 2-MSH.

Different cardiovascular profiles of three melanocortins in conscious rats; evidence for antagonism between gamma 2-MSH and ACTH-(1-24).

1. We investigated the effects of [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), adrenocorticotropin-(1-24) (ACTH-(1-24)) and gamma 2-MSH, three melanocortins with different agonist selectivity for the five cloned melanocortin receptors, on blood pressure and heart rate in conscious, freely moving rats following intravenous administration. 2. As was previously found by other investigators as well as by us gamma 2-MSH, a peptide suggested to be an agonist with selectivity for the melanocortin MC3 receptor, caused a dose-dependent, short lasting pressor response in combination with a tachycardia. Despite the fact that NDP-MSH is a potent agonist of various melanocortin receptor subtypes, among which the melanocortin MC1 receptor, it did not affect blood pressure or heart rate, when administered i.v. in doses of up to 1000 nmol kg-1. 3. ACTH-(1-24) caused a dose-dependent decrease in blood pressure in combination with a dose-dependent increase in heart rate in a dose-range from 15 to 500 nmol kg-1. The cardiovascular effects of ACTH-(1-24) were independent of the presence of the adrenals. 4. Pretreatment with ACTH-(1-24) caused a pronounced, dose-dependent parallel shift to the right of the dose-response curve for the pressor and tachycardiac effects of gamma 2-MSH. The antagonistic effect of ACTH-(1-24) was already apparent following a dose of this peptide as low as 10 nmol kg-1, which when given alone had no intrinsic hypotensive activity. 5. These results form further support for the notion that it is not via activation of one of the as yet cloned melanocortin receptors that gamma-MSH-like peptides increase blood pressure and heart rate. The cardiovascular effects of ACTH-(1-24) seem not to be mediated by the adrenal melanocortin MC3 receptors, for which ACTH-(1-24) is a selective agonist, or by adrenal catecholamines. 6. There appears to be a functional antagonism between ACTH-(1-24) and gamma 2-MSH, two melanocortins derived from a common precursor, with respect to their effect on blood pressure and heart rate. Whether this antagonism plays a (patho)physiological role remains to be shown.

Structure-activity analysis for the effects of gamma-MSH/ACTH-like peptides on cerebral hemodynamics in rats.

In a previous structure-activity analysis we have shown that the gamma-melanocyte-stimulating hormones (gamma-MSHs) and structurally related adrenocorticotropic hormone (ACTH) fragments share an amino-acid sequence which is determinant for the effects of these peptides on peripheral hemodynamics, viz. a pressor and a tachycardiac response, in conscious rats. We now investigated whether these structural features are also important for the effects of these peptides on cerebral hemodynamics in urethane-anesthetized rats. After intracarotid and intravenous administration, the 'mother' peptides, Lys-gamma2-MSH and gamma2-MSH, and, with a 10-fold lower potency, ACTH-(4-10), caused a dose-dependent pressor and tachycardiac response, as well as an increase in extra- and intracranial blood flow and microcirculatory cerebrocortical blood flow. Removal of C-terminal amino acids resulted in gamma-MSH-fragments which were devoid of effects on peripheral and central hemodynamics. Fragments of gamma2-MSH which were shortened at the N-terminal side (gamma-MSH-(4-12) and gamma-MSH-(5-12)) were less potent than gamma2-MSH, but had an intrinsic activity similar to that of gamma2-MSH with respect to the pressor and tachycardiac effect. However, the potency and intrinsic activity of these shortened fragments on intracerebral hemodynamic parameters were the same as those of gamma2-MSH. This suggests that different mechanisms (e.g., site of action and/or melanocortin receptor subtype) are involved in the cerebral hemodynamic effects of the melanocortins and in their peripheral hemodynamic effects. Surprisingly, removal of an additional residue, His5, resulting in the fragment gamma-MSH-(6-12), led to full restoration of potency with respect to extracranial blood flow, blood pressure and heart rate. Neither the structurally related analog, [Nle4,D-Phe7]alpha-MSH (NDP-MSH), nor ACTH-(1-24) was able to induce a pressor effect or cerebral hemodynamic effects. In contrast, both compounds had a depressor effect. It is concluded that the C-terminal amino acids in the structure of gamma-MSH/ACTH-like peptides are essential for efficacy for the central hemodynamic effects, i.e., the increase in intracerebral (microcirculatory) blood flow. However, in contrast to what holds for the peripheral hemodynamic features, the N-terminal sequence has hardly any influence on potency or efficacy. The results with NDP-MSH and ACTH-(1-24) and the other fragments lead us to postulate that it is not one of the five known subtypes of melanocortin receptors which mediates the hemodynamic effects of the melanocortins, but an additional, still unidentified subtype. A clue for the elucidation of such a receptor might be found in the structural features of gamma-MSH-(6-12) that appear to be very important determinants for the effectiveness to alter peripheral and central hemodynamics.

Distribution of Lys-gamma 2-melanocyte-stimulating hormone- (Lys-gamma 2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat.

Using an antiserum raised against Lys- gamma 2-melanocyte-stimulating hormone (Lys- gamma 2-MSH), with a high specificity for this peptide and its des-Lys derivative, gamma 2-MSH, we found Lys- gamma 2-MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain region containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-gamma 2-MSH-like immunoreactivity is considerably more widespread than that of mRNA for the 'gamma-MSH receptor' (the melanocortin MC3 receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-gamma 2-MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-gamma 2-MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of gamma-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-gamma 2-MSH-containing fibers suggests a role of the gamma-MSHs in cardiac function.

The effects of gamma 2-melanocyte-stimulating hormone and nimodipine on cortical blood flow and infarction volume in two rat models of middle cerebral artery occlusion.

We observed that the pro-opiomelanocortin-derived neuropeptide, gamma 2-melanocyte-stimulating hormone (gamma 2-MSH), has various peripheral and central hemodynamic effects in the rat, including a marked enhancing effect on cerebral blood flow. This hemodynamic profile might be of interest in the pharmacotherapeutic approach to acute cerebral ischemia. Being an adrenocorticotropin (ACTH) analogue, gamma 2-MSH might also possess direct neuronal protective properties. Therefore, in two rat models of focal cerebral ischemia we studied the effects of gamma 2-MSH, with nimodipine, a Ca2+ channel antagonist, as a reference compound, on parasagittal laser-Doppler-assessed cortical blood flow and infarction volume. In isoflurane-anesthetized Wistar and F344 rats i.v. bolus infusions (four in total) of gamma 2-MSH or nimodipine or their vehicle controls were given 1 h before, 1 min after, and 1 h and 2 h after occlusion of the middle cerebral artery. We used both an intravasal and an extravasal middle cerebral artery occlusion technique because pilot experiments had shown differences in the severity of ischemia with the two techniques. gamma 2-MSH (100 nmol/kg in 1 min) increased cortical blood flow significantly but transiently, both pre- and post-ischemically, whereas nimodipine (20 micrograms/kg in 1 min) increased cortical blood flow only pre-ischemically in both models of middle cerebral artery occlusion. gamma 2-MSH had no effect on cortical and striatal infarction volume, while nimodipine caused a significant reduction of cortical infarction volume in the extravasal middle cerebral artery occlusion model. To conclude, despite its hemodynamic and possible neuroprotective properties, gamma 2-MSH did not prevent ischemic neuronal damage after middle cerebral artery occlusion in rats. This might be partly due to the short half-life of the peptide, leading to a transient increase in cortical blood flow and short neuronal exposure time, suggesting that prolonged infusion of the neuropeptide might be required. The results with nimodipine support the notion that it attenuates cortical ischemic damage, independently of effects on cerebral hemodynamics.

Differences in striatal extracellular amino acid concentrations between Wistar and Fischer 344 rats after middle cerebral artery occlusion.

We hypothesized that the interstrain difference between Wistar and Fischer-344 (F344) rats in cerebral infarction volume after proximal middle cerebral artery (MCA) occlusion might be explained by differences in excitotoxicity between both rat strains. Using microdialysis we measured during a 5 h period after MCA occlusion the release of aspartate, glutamate and taurine in the cerebral cortex and the striatum. The volume of striatal infarction was comparable in Wistar and F344 rats. We found, however, in Wistar rats a significantly higher striatal efflux of aspartate and glutamate than in F344 rats, whereas the striatal taurine efflux was of a similar magnitude in the two strains. Because of the (variably) smaller volume of cortical infarction in Wistar rats (than that in F344), the location of the microdialysis probe-membrane with respect to the area of cortical infarction differed between Wistar rats. Hence, a reliable comparison between the quantitative amount of amino acids in the dialysate from the cortical probes of both rat strains could not be made. These results, demonstrating differences in striatal excitotoxicity between Wistar and F344 rats after MCA occlusion, are the first to show interstrain differences in striatal pathophysiology of focal ischemia between these normotensive rat strains. They do however not explain why MCA occlusion results in a significantly different volume of cortical infarction between Wistar and F344 rats. The F344 strain will probably show in a more sensitive way, as compared to Wistar rats, neuroprotective effects of agents that diminish excitotoxic damage during focal cerebral ischemia.

Middle cerebral artery occlusion in Wistar and Fischer-344 rats: functional and morphological assessment of the model.

Cerebral infarction volume after occlusion of a short proximal segment of the middle cerebral artery (MCA) is reported to be different in Wistar compared to Fischer-344 (F344) rats, in both size and variability. Knowledge about the cause of these differences might enable us to explain and perhaps reduce the variation in infarct volume and create a reproducible model of focal cerebral ischemia in the rat. We investigated in Wistar and F344 rats both the effect of occlusion of a long proximal MCA segment on cerebral infarction volume, visualized by magnetic resonance imaging and histology, and the morphology of the major cerebral arteries. Occlusion of a long proximal MCA segment resulted in a striatal and a small cortical infarction in Wistar and a striatal and sizable cortical infarction in F344 rats (as is the case after occlusion of a short proximal MCA segment). In Wistar rats, however, occlusion of a long proximal MCA segment strongly reduced the variability in infarction volume in comparison to occlusion of a small proximal MCA segment. Analysis of the morphology of the major cerebral arteries showed a significantly higher number of proximal side branches of the long proximal MCA segment in Wistar rates than in F344 rats. We conclude that after short-segment proximal MCA occlusion, extreme variability in cerebral infarction volume in Wistar rats compared to F344 rats may be attributable to a significantly greater number of proximal MCA side branches in Wistar rats than in F344 rats.

Cardiovascular effects of gamma-MSH/ACTH-like peptides: structure-activity relationship.

Intravenous administration of gamma2-melanocyte-stimulating hormone (gamma2-MSH) to conscious rats causes a dose-dependent increase in blood pressure and heart rate, while the structurally related peptide adrenocorticotropic hormone-(4-10) (ACTH-(4-10)) is 5-10 times less potent in this respect. This prompted us to investigate which amino acid sequence is determinant for the cardiovascular selectivity of peptides of the gamma-MSH family. Lys-gamma2-MSH, most likely the endogenously occurring gamma-MSH analog, was as potent as gamma2-MSH in inducing increases in blood pressure and heart rate. Removal of C-terminal amino acids resulted in gamma-MSH-fragments which were devoid of cardiovascular activities. Removal of amino acids from the N-terminal side of gamma2-MSH resulted in fragments which were less potent, but had an intrinsic activity not different from that of gamma-MSH. Surprisingly, gamma-MSH-(6-12) was more potent than gamma2-MSH. The shortest fragment which displayed pressor and tachycardiac responses was the MSH 'core', His-Phe-Arg-Trp (= gamma-MSH-(5-8)), which is identical to ACTH-(6-9). This was corroborated by testing fragments of ACTH-(4-10). We conclude that the message essential for cardiovascular effects resides in the gamma-MSH-(5-8)/ACTH-(6-9) sequence. Proper C-terminal elongation is required for full expression of cardiovascular activity of gamma2-MSH, as the sequence of Asp9-Arg10-Phe11 appears to play an important role in establishing intrinsic activity. The amino acids N-terminal to the MSH 'core' sequence appear to be essential for the potency of the peptides.

4-Aminopyridine differentially affects the spontaneous release of radiolabelled transmitters from rat brain slices in vitro.

4-Aminopyridine increased the release of [3H]noradrenaline from dorsal hippocampus slices in vitro in a concentration-dependent manner. When the slices were exposed to 4-aminopyridine for 5 min, the overflow of radioactivity returned to pre-exposure values within 20-25 min. When the exposure of the slices was continued, a sustained enhancement of the release of [3H]noradrenaline was observed for the duration of the exposure. 4-Aminopyridine, 10(-4) M, had an effect of similar magnitude, or an even more pronounced effect, on the release of [3H]catecholamine from cortex, septum, periaqueductal gray and striatum slices. The effects of the compound on the release of [3H]5-hydroxytryptamine and [14C]acetylcholine were less pronounced. At this concentration 4-aminopyridine had no effect on the release of [3H]D-aspartate from hippocampus or septum slices, whereas the effect on the release of this transmitter in striatal slices was marginal. The effect of 4-aminopyridine on the release of [3H]noradrenaline in hippocampus slices was largely dependent on the presence of Ca2+ in the superfusion medium. This was also the case for the effect on the release of [3H]noradrenaline from preloaded dorsal hippocampus synaptosomes. In the presence of nitrendipine the effect of 4-aminopyridine was dose-dependently reduced, but the maximal reduction, at a nitrendipine concentration of 10(-4) M, was only 40%. Cd2+ completely abolished the effect of 4-aminopyridine on the release of [3H]noradrenaline. These results confirm that the enhancing effect of 4-aminopyridine on the release of [3H]noradrenaline depends on the entry of extracellular Ca2+ into the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of gamma 2-melanocyte-stimulating hormone on cerebral blood flow in rats.

The effects of the proopiomelanocortin-(POMC)-derived peptide gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) on mean arterial blood pressure (BP: MAP), heart rate (HR), internal and total carotid blood flow (BF) (CFint and CFtot, respectively), and regional cerebrocortical blood flow (CBF) were measured in urethane-anesthetized rats after intravenous (i.v.) and intracarotid (i.car.) administration of the peptide. gamma 2-MSH (1.5-100 nmol/kg) administered i.v. and i.car. caused a dose-dependent increase in MAP and HR. Injection of the peptide i.car. in the middose range resulted in a more pronounced pressor effect. Furthermore, the earlier onset of the hemodynamic effects after i.car. injection suggests that forebrain structures play a role in these effects. In addition to the pressor response, gamma 2-MSH produced a strong increase in CFint, CFtot, and CBF after both routes of administration, suggesting an increased intracerebral BF. Whereas the effects of the higher doses of gamma 2-MSH on MAP and CFtot were quantitatively comparable after either the intravenous or intracarotid administration, the effect on regional CBF and CFint was about twice as high after i.car. infusion, indicating a centrally mediated phenomenon underlying this effect on CBF. The increase in CFint cannot in itself be ascribed to a gamma 2-MSH-mediated higher perfusion pressure (i.e., BP), since an equipressor dose of norepinephrine (NE) caused a significant decrease in CFint. The significant and more than twofold higher increase in CBF after intracarotid administration of gamma 2-MSH in comparison with administration of NE by the same route also suggests a central origin for the enhancement of microcirculatory flow due to the peptide.

The nigrostriatal dopamine system: role in the development of hypertension in spontaneously hypertensive rats.

The effects of lesions in the pars compacta of the substantia nigra of 4-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied on (a) changes in blood pressure during a period of 6 weeks following lesioning, and (b) dopamine and metabolite concentrations in the caudate nucleus and (c) the in vitro release of [3H]dopamine from caudate nucleus slices 6 weeks after the lesioning. We report here that substantia nigra lesions caused a profound attenuation of the development of hypertension in SHR. No effect on heart rate was observed. Whereas the concentrations of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the caudate nucleus were not different, the amount of homovanillic acid (HVA) and the ratios DOPAC/dopamine and HVA/dopamine were lower in sham-treated SHR than in sham-treated WKY, indicating a lower turnover of dopamine in SHR. Six weeks after lesioning, the concentrations of dopamine, DOPAC and HVA were decreased in the caudate nucleus (46-66%) of both strains, whereas DOPAC/dopamine and HVA/dopamine ratios tended to be increased in both SHR and WKY. Using an in vitro superfusion method, it was found, that the electrically stimulated release of [3H]dopamine from caudate nucleus slices of sham-treated SHR was lower than from slices of sham-treated WKY at the age of 10 weeks. Six weeks after lesioning, the stimulus-evoked release of [3H]dopamine from the remaining terminals was significantly increased in caudate nucleus slices of SHR, whereas the difference did not reach statistical significance in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

A hypotensive and bradycardic action of gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) microinjected into the nucleus tractus solitarii of the rat.

In conscious and urethane-anesthetized rats intravenously (i.v.) administered gamma 2-melanocyte-stimulating hormone (gamma 2-MSH), a melanotropin derived from the precursor peptide pro-opiomelanocortin (POMC), has been shown to induce a pressor response combined with a tachycardia. A site of action within the hindbrain, e.g. the nucleus tractus solitarii (NTS) or the area postrema (AP), has been suggested. In order to test the postulate that gamma 2-MSH acts within these hindbrain regions, the peptide was microinjected into various parts of the NTS and into the AP of urethane-anesthetized rats and blood pressure (BP) and heart rate (HR) were measured. Injection of gamma 2-MSH (100-500 pmol) into the NTS resulted in a dose-dependent decrease in BP and HR rather than in the expected pressor and tachycardiac response which is generally found in conscious and urethane-anesthetized rats following i.v. administration of the peptide. With respect to the depressor and bradycardiac effect the melanotropin was far more potent when injected into the pars commissuralis than into the medial part of the NTS. The responses were maximal after 3-4 min and lasted for about 15 min. gamma 2-MSH had no effect when injected into the AP. It is noteworthy that also a hypotensive and bradycardic effect for gamma 2-MSH is found in pentobarbital-anesthetized rats following i.v. administration. Therefore, we conclude that in addition to a pressor and tachycardic response gamma 2-MSH can elicit an opposite effect by interaction with structures within a discrete region in the NTS, the pars commissuralis.(ABSTRACT TRUNCATED AT 250 WORDS)

Synchronism of pressor response and grooming behavior in freely moving, conscious rats following intracerebroventricular administration of ACTH/MSH-like peptides.

After the i.c.v. administration of 300 pmol ACTH-(1-24) or [Nle4,D-Phe7]alpha-MSH, a long-lasting increase in blood pressure was observed synchronously with the incidence of excessive grooming. Two structurally related peptides with no grooming behavior-inducing potency, ACTH-(7-16)-NH2 and gamma 2-MSH, in doses of 300 and 500 pmol, respectively, caused a slight and short-lasting increase in blood pressure or had no effect, respectively. When the grooming behavior-inducing effect of ACTH-(1-24) was abolished, either by the prior manipulation of central dopaminergic neurotransmission by the i.c.v. administration of the dopamine receptor antagonist, haloperidol, or, due to the occurrence of single-dose tolerance to ACTH-(1-24), the pressor response was abolished as well. These data are in support of the postulate that the incidence of grooming behavior and the elevation of blood pressure are temporally associated and indicate that the two phenomena are causally related.

Peptide-induced grooming behavior and caudate nucleus dopamine release.

We simultaneously measured the display of grooming behavior and, by monitoring the extracellular dopamine concentration via transversal microdialysis, the release of dopamine in the caudate nucleus in freely moving rats after i.c.v. administration of 1 micrograms adrenocorticotropic hormone-(1-24) (ACTH-(1-24)). During a period of 1 h after administration of the peptide, the incidence of excessive grooming behavior was increased. Concomitantly, the concentration of dopamine in the caudate nucleus dialysates was significantly increased (maximal effect 151% of basal release) whereas that of its metabolite DOPAC was unchanged. The potent alpha-melanocyte stimulating hormone (alpha-MSH) receptor agonist, [Nle4,D-Phe7]alpha-MSH, induced grooming behavior and stimulated caudate nucleus dopamine release (maximal effect 148% of basal release) whereas ACTH-(7-16)-NH2 did neither induce grooming behavior nor cause an increase in caudate nucleus dopamine release. Single-dose tolerance was observed for ACTH-induced grooming but not for ACTH-induced dopamine release. These data are in support of the proposed involvement of brain dopamine systems in grooming behavior of the rat but at the same time suggest that the effect of ACTH/MSH-like peptides on dopaminergic transmission in the caudate nucleus is proximal to the final neural pathway involved in ACTH-induced grooming behavior.