Carina M. Kasbergen

The hemodynamic effects of γ2-melanocyte-stimulating hormone and related melanotropins depend on the arousal potential of the rat

In conscious rats, i.v. administered adrenocorticotropic hormone (ACTH-(4-10)) and gamma 2-melanocyte-stimulating hormone (gamma 2-MSH) induced a dose-dependent increase in blood pressure (BP), heart rate (HR) and pulse pressure (PP). No circadian influence on these effects was observed. The structurally related peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), only caused an increase in HR, which was not dose-dependent, whereas the stable ACTH-(4-9) analog, Org 2766, was without effect on these hemodynamic parameters. In rats under light urethane-induced anesthesia, which is known to maintain reflexes and sufficient sympathetic tone, gamma 2-MSH caused hemodynamic responses similar to those observed in conscious rats. In contrast, gamma 2-MSH had an opposite effect in rats under deep pentobarbital-induced anesthesia: a depressor effect combined with a slight bradycardia. A comparative study with rats of a more arousable Wistar rat substrain (Riv:TOX) and of a less excitable rat substrain (U:WU) showed that the dose-pressor response curves for ACTH-(4-10) and gamma 2-MSH were shifted to the left in the more excitable rats as compared to the in the less excitable rats. We conclude that a restricted amino acid sequence in the N-terminal part of the pro-opiomelanocortin (POMC)-molecule (gamma 2-MSH/ACTH-(4-10)-like) is responsible for the stimulating effects on the cardiovascular system and that those effects are strongly dependent on the state of arousal, i.e. sympathetic tone, of the rat. These stimulatory effects override a depressor phenomenon which can only be detected during central depression.

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.

Relevance of the C‐terminal Arg‐Phe sequence in γ2‐melanocyte‐stimulating hormone (γ2‐MSH) for inducing cardiovascular effects in conscious rats

The cardiovascular effects by γ2‐melanocyte‐stimulating hormone (γ2‐MSH) are probably not due to any of the well‐known melanocortin subtype receptors. We hypothesize that the receptor for Phe‐Met‐Arg‐Phe‐amide (FMRFa) or Phe‐Leu‐Phe‐Gln‐Pro‐Gln‐Arg‐Phe‐amide (neuropeptide FF; NPFFa), other Arg‐Phe containing peptides, is the candidate receptor. Therefore, we studied various Arg‐Phe containing peptides to compare their haemodynamic profile with that of γ2‐MSH(6–12), the most potent fragment of γ2‐MSH. Mean arterial pressure (MAP) and heart rate (HR) changes were measured in conscious rats after intravenous administration of γ2‐MSH related peptides. Phe‐Arg‐Trp‐Asp‐Arg‐Phe‐Gly (γ2‐MSH(6–12)), FMRFa, NPFFa, Met‐enkephalin‐Arg‐Phe‐amide (MERFa), Arg‐Phe‐amide (RFa), acetyl‐Phe‐norLeu‐Arg‐Phe‐amide (acFnLRFa) and desamino‐Tyr‐Phe‐norLeu‐Arg‐Phe‐amide (daYFnLRFa) caused a dose‐dependent increase in MAP and HR. γ2‐MSH(6–12) showed the most potent cardiovascular effects (ED50=12 nmol kg−1 for ΔMAP; 7 nmol kg−1 for ΔHR), as compared to the other Arg‐Phe containing peptides (ED50=177–292 nmol kg−1 for ΔMAP; 130–260 nmol kg−1 for ΔHR). Peptides, which lack the C‐terminal Arg‐Phe sequence (Lys‐Tyr‐Val‐Met‐Gly‐His‐Phe‐Arg‐Trp‐Asp‐Arg‐Pro‐Gly (γ2‐pro11‐MSH), desamino‐Tyr‐Phe‐norLeu‐Arg‐[L‐1,2,3,4 tetrahydroisoquinoline‐3‐carboxylic acid]‐amide (daYFnLR[TIC]a) and Met‐enkephalin (ME)), were devoid of cardiovascular actions. The results indicate that the baroreceptor reflex‐mediated reduction of tonic sympathetic activity due to pressor effects is inhibited by γ2‐MSH(6–12) and that its cardiovascular effects are dependent on the presence of a C‐terminal Arg‐Phe sequence. It is suggested that the FMRFa/NPFFa receptor is the likely candidate receptor, involved in these cardiovascular effects.

Presynaptic deficit of sympathetic nerves: a cause for disturbed sciatic nerve blood flow responsiveness in diabetic rats

Reduced nerve blood flow is thought to play an important role in the pathogenesis of diabetic neuropathy. This disturbance in nerve blood flow might be the consequence of either microangiopathy or an impaired autonomic innervation of the vasa nervorum. In order to differentiate between a vascular or an adrenergic-autonomic defect as the underlying cause of the disturbed nerve blood flow, we investigated the effects of the adrenocorticotropic hormone [ACTH]-(4-9) analogue Org 2766 on sciatic nerve blood flow under basal and adrenergic-stimulated conditions. Org 2766 has neuroprotective effects without cardiovascular effects. Treatment with Org 2766 was started 6 weeks after the induction of experimental diabetes mellitus. At week 12 the sciatic nerve blood flow, measured by laser-Doppler flowmetry, was reduced to 60% of the non-diabetic level; blood pressure was unchanged in diabetic rats compared to non-diabetic rats. Basal haemodynamic values were not affected by Org 2766 treatment. Vasa nervorum adrenergic responsiveness to tyramine (presynaptic) and phenylephrine (postsynaptic) was investigated. Diabetic rats showed adrenergic hyporesponsiveness. Treatment with Org 2766 restored the reduced presynaptic response to tyramine without affecting the reduced postsynaptic response to phenylephrine. It is concluded that a presynaptic-sympathetic deficit of nervi vasorum causes a disturbed flow responsiveness in diabetic rat sciatic nerve and that adrenergic autonomic disturbances in the vasa nervorum have only a small role in the reduced basal nerve blood flow of diabetic rats.

In vivo cardiovascular reactivity and baroreflex activity in diabetic rats

OBJECTIVES Abnormalities of the cardiovascular system, e.g. impaired vasoreactivity and changes in baroreflex control of heart rate, are known to occur in experimental diabetes. It is not clear whether these cardiovascular dysfunctions are direct consequences of cardiovascular deficits and/or have autonomic neuropathy as a cause. METHODS To differentiate between cardiovascular deficits or neuronal impairment as a cause for these cardiovascular dysfunctions, we tested the effects of the ACTH4-9 analogue, Org 2766, a neurotrophic compound without cardiovascular effects, on arterial pressure, heart rate and baroreflex control of heart rate. At 15 weeks, rats were made diabetic by injection of streptozotocin, and from 0-6, 6-12 or 12-18 weeks thereafter 3 groups of rats were treated with Org 2766. These effects were evaluated during phenylephrine-induced increases, and sodium nitroprusside-induced decreases, in blood pressure, in rats that had been diabetic for various periods (2-42 weeks). RESULTS Throughout, both depressor response and maximal vasodilator activity in response to sodium nitroprusside were significantly (P < 0.05) reduced as compared to those of the non-diabetic controls. The pressor response of the diabetic rats to phenylephrine was only significantly (P < 0.05) reduced at 4, 6 and 12 weeks, and at 18 weeks, the diabetic rats were either hypo- or normoresponsive; Org 2766 did not restore the disturbed pressor response. From weeks 4 to 42 both maximal decrease in heart rate and sensitivity of baroreflex-mediated bradycardia in the diabetic rats were significantly less (P < 0.05) than those in the non-diabetic controls. Org 2766 restored the diminished baroreflex-mediated bradycardia of diabetic rats to non-diabetic control levels at 6 weeks, had an ameliorating effect at 12 weeks and no effect at 18 weeks. CONCLUSIONS Time-dependent decreases in baroreflex sensitivity in diabetic rats was demonstrated and a much less steep decline of baroreflex sensitivity occurred in non-diabetic control rats. The ACTH4-9 analogue, Org 2766, when given immediately upon the induction of diabetes seem to delay the development of autonomic neuropathy, which suggests that cardiovascular factors appear to be of minor importance.

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.

A disturbed macrocirculatory supply as a determinant for a reduced sciatic nerve blood flow in diabetic rats

The aim of this study was to evaluate macrocirculatory disturbances in relation to the reduced sciatic nerve blood flow seen in diabetic rats. Therefore, both femoral blood flow, the macrocirculatory arterial blood supply to the sciatic nerve, and the microcirculatory neuronal blood flow were measured. In order to differentiate between a direct vascular or a neuronal defect as a cause for the disturbed macrocirculatory blood flow the effects of the adrenocorticotropic hormone [ACTH]-(4-9) analogue, Org 2766, a neurotrophic compound without cardiovascular effects, were investigated on the femoral flow under basal as well as adrenergic-stimulated conditions. Adrenergic responsiveness to tyramine and phenylephrine effect on femoral flow was determined. Basal sciatic nerve and femoral blood flow were reduced by 48% and 42%, respectively, after 12 weeks of diabetes, without effect on blood pressure. Treatment with Org 2766, beginning 6 weeks after the induction of diabetes, had no influence on these basal haemodynamic variables. Femoral flow in diabetic rats showed a smaller response to tyramine and phenylephrine compared to the control. Org 2766 restored this disturbed flow response to that of the control rats. In conclusion, the decrease in basal femoral flow might be responsible for the lowered sciatic nerve blood flow. Although neuronal disturbances due to diabetes had a very minor role in the reduction of basal femoral blood flow the adrenergic-stimulated flow responsiveness was seriously affected in diabetic rats.

Effect of γ-melanocyte-stimulating hormones on baroreflex sensitivity and cerebral blood flow autoregulation in rats

OBJECTIVE In the present paper, we are interested in the effects of gamma-melanocyte-stimulating hormones (gamma-MSHs) on cardiovascular regulatory systems. METHODS Mean arterial pressure (MAP), cerebral blood flow (CBF) and heart rate (HR) were measured in urethane-anaesthetised rats after intravenous administration of lysgamma(2)-MSH, gamma(2)-MSH, gamma(2)-MSH(6-12) or phenylephrine. RESULTS The gamma-MSHs caused an increase in MAP, CBF and HR, whereas phenylephrine caused an increase in MAP and CBF and baroreceptor reflex-mediated bradycardia. All tested gamma-MSHs showed a significant impairment of the baroreceptor reflex sensitivity and CBF autoregulation as compared to the phenylephrine group. gamma(2)-MSH shows identical effects on the baroreceptor reflex and CBF as the endogenous occurring lysgamma(2)-MSH. In addition, the C-terminal fragment of gamma(2)-MSH, gamma(2)-MSH(6-12), induced similar effects as gamma(2)-MSH. The level of increase in MAP was comparable between the gamma-MSHs and the phenylephrine group. CONCLUSIONS The present study suggests that gamma(2)-MSH and the shorter fragment gamma(2)-MSH(6-12) impair baroreceptor reflex sensitivity, due to a strong increase in sympathetic tone and/or change in baroreceptor reflex setpoint, and induce cerebrovasodilatation, which can counteract an autoregulation-mediated cerebrovasoconstriction due to systemic pressor effects. Furthermore, the results indicate that the C-terminal site of gamma(2)-MSH is relevant for its central-mediated inhibitory effects on the baroreceptor reflex and CBF.