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

Abstract

1. 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.

2. Mean arterial pressure (MAP) and heart rate (HR) changes were measured in conscious rats after intravenous administration of γ₂-MSH related peptides.

3. 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).

4. 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.

5. 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.

6. It is suggested that the FMRFa/NPFFa receptor is the likely candidate receptor, involved in these cardiovascular effects.

Introduction

Melanocortins (α-melanocyte-stimulating hormone (α-MSH), β-MSH, γ-MSH and adrenocorticotropic hormone (ACTH)) belong to a family of peptides derived from the precursor pro-opiomelanocortin (see Table 1). In addition to their effects on the hormonal system, behaviour, temperature and feeding regulation, γ-MSHs and in particular γ₂-MSH and its shorter fragment γ₂-MSH(6–12) have strong effects on the cardiovascular system (Callahan et al., 1985; 1988; Li et al., 1996; Sun et al., 1992; Van Bergen et al., 1995; 1997a). Systemic administration of γ₂-MSH and γ₂-MSH(6–12) to rats causes a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). So far, a most likely candidate receptor, involved in these cardiovascular effects, is the melanocortin (MC) receptor. There are indications for a centrally located target for γ-MSH (Sun et al., 1992; Van Bergen et al., 1998; Fodor et al., 1996). As the MC3-4 receptor subtypes are mainly expressed in the brain (Gantz et al., 1993; Mountjoy et al., 1994), these subtypes are most likely involved in γ-MSH-induced cardiovascular actions. However, there are results that are in contradiction with this hypothesis. α-MSH shows high affinity for and can activate all subtype MC receptors (Adan et al., 1994; Gantz et al., 1994; Mountjoy, 1994), but systemic administration of this peptide has no influence on the cardiovascular system (Van Bergen et al., 1997b). Furthermore, γ₂-MSH(6–12) shows very potent pressor and cardioaccelerator effects (Van Bergen et al., 1995), but has no affinity and cannot activate any of the brain MC receptor subtypes in vitro (Oosterom, unpublished observations). These studies indicate that the cardiovascular effects of γ-MSH's are probably not due to either of the well-known MC subtype receptors.

Table 1.

Amino acid sequence of various melanocortins and FMRFa-like peptides

The molluscan peptide Phe-Met-Arg-Phe-amide (FMRFa) and its mammalian analogues Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide (Met-enkephalin-Arg-Phe-amide; MERFa) and Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide (neuropeptide FF; NPFFa) were isolated from bovine, rat and human central nervous system (Majane et al., 1983; Tang et al., 1984; Yang et al., 1983; 1985). The chemical structural resemblance (Arg-Phe-amide) between FMRFa-like peptides and γ-MSHs, their comparable haemodynamic profile (increase in MAP and HR) (Allard et al., 1995; Barnard & Dockray, 1984a; De Wildt et al., 1993; Mues et al., 1982; Thiemermann et al., 1991; Wong et al., 1985) and correspondent anatomical location of these peptides and their receptors within the brain (hypothalamus and nucleus tractus solitarius) (Fodor et al., 1996; Kivipelto et al., 1989; 1992; Majane et al., 1989; O'Donohue et al., 1984; Pittius et al., 1984), makes the FMRFa/NPFFa/MERFa receptor a very important candidate for mediating the cardiovascular effects of γ-MSHs. Therefore, in the present study various peptides with a C-terminal Arg-Phe structure are tested for their haemodynamic profile in conscious rats. Furthermore, peptides with C-terminal modifications are used to indicate that the C-terminal Arg-Phe structure is indeed essential for bioactivity.

The goal of this study is to examine which structure of γ₂-MSH(6–12), the most potent fragment of γ₂-MSH, is responsible for the cardiovascular effects. This knowledge may lead to a suggestion for a candidate receptor and its related mechanism of action responsible for the cardiovascular effects of γ₂-MSH(6–12).

Methods

Animals

Naive male albino Wistar rats (U:WU/CPB) weighing 230–280 g were used. Two or three rats were housed per Macrolon cage (23×17×14 cm) containing a layer of woodshavings under conditions of constant ambient temperature (21±1°C), constant humidity (60±15%), and light/dark rhythm (with lights on from 0700 to 1900 h). After surgery, the animals were housed individually in Plexiglas cages (25×25×25 cm) under presurgical conditions. Food (complete laboratory chow: Hope Farms, Woerden, The Netherlands) and water were accessible ad libitum throughout the experiment.

Surgery

Rats were anaesthetized with halothane (O₂/NO₂ 1 : 2; introduction 5%; maintenance 1.3–1.7%; Fluothane®, Zeneca BV, Ridderkerk, The Netherlands). Prior to operation each rat received eye ointment (Caf⁺, Alpharmo BV, Arnhem, The Netherlands) to protect the eyes against dehydration. During the operation, body temperature of the rats was maintained with a heated pad (K-temp, I.M.S., Helmond, The Netherlands).

For BP and HR measurements, the femoral artery was cannulated with a polyethylene tubing (PE10, i.d. 0.28 mm, o.d. 0.61 mm; Portex, The Hague, The Netherlands), which was melted to another polyethylene tubing (PE50, i.d. 0.58 mm, o.d. 0.96 mm; Portex) with a 180° loop. The latter was melted to third polyethylene tubing (PE100, i.d. 0.86 mm, o.d. 1.52 mm; Portex). The cannula was filled with a heparin solution (50 IU ml⁻¹, Leo Pharmaceutical Products BV, Weesp, The Netherlands) and guided underneath the skin to the head. For i.v. administration of drugs the jugular vein was cannulated according to Steffens (1969). The silastic cannula (i.d. 0.51 mm, o.d. 0.94 mm, Dispo Medical BV, Hattum, The Netherlands) was filled with saline and guided underneath the skin towards the head. Both cannulae were connected to a steel connector with a 90° loop, which in turn was connected to a short polyethylene tubing and closed by a steel stylet. Both cannulae were fixated to the skull with dental cement (Dental Union BV, Nieuwegein, The Netherlands). After the operation the rats received a single injection of saline (1.5 ml, subcutaneously (s.c.)) and were allowed to recover until they regained consciousness in a heated chamber (I.M.S.).

Cardiovascular measurements and intravenous administration

Forty-five min prior to baseline recordings of BP and HR, rats were connected to long (0.5 m) polyethylene tubings (Portex) for stress-free cardiovascular measurements and i.v. administration. The arterial cannula was connected to a pressure transducer (Viggo-Spectramed, disposable DTX/plus, Ohmeda, Bilthoven, The Netherlands) by a PE100 tubing (i.d. 0.86 mm, o.d. 1.52 mm; Portex). The pressure transducer was connected to a DC-preamplifier and biotachometer (Instrument service, Utrecht University, The Netherlands) coupled to a P75-computer. Data were continuously recorded and measured with the HDAS (Haemodynamic Data Acquisition System) (Instrumental Department, University of Limburg, Maastricht, The Netherlands) and DatView program (Instrumental Department, University of Limburg, Maastricht, The Netherlands). Mean arterial pressure (MAP) was calculated according to the formula: (2×P_d+P_s)/3, in which P_d is diastolic pressure and P_s systolic pressure. The jugular vein cannula was attached to a syringe (1000 μl, Inacom Instruments, Veenendaal, The Netherlands) and microinfusion pump (Harvard Inc., Massachusetts, U.S.A.) by a PE50 tube (i.d. 0.58 mm, o.d. 0.96 mm, Portex). Systemic injections were performed at 500 μl min⁻¹.

Experimental design

Rats were surgically equipped with a femoral artery and jugular vein cannula under sterile conditions for measurements of blood pressure (BP) and heart rate (HR) and for intravenous (i.v.) administration of drugs, respectively. They were allowed to recover from surgery for 3 days. During the recovery period, the animals were handled daily for weighing and habituation purposes. Cardiovascular measurements and i.v. injections were performed stressfree in conscious, resting rats by a long-line technique. Baseline BP and HR were recorded before i.v. injections. Subsequently, rats were injected i.v. with phenylephrine (5 μg kg⁻¹) to check whether the animals show a significant increase in BP. All rats responded and were used for further measurements. Before drug treatment, rats were injected with saline (200 μl). One drug was injected per rat in a volume of 100 μl and an interval of 15 min was used between each dose of injection to allow stabilization of BP and HR. In order to flush the cannula each infusion of a drug was followed by 100 μl saline.

All experiments were performed in the home cage during the light phase of the circadian cycle between 0900 and 1400 h. After the experiment, all rats were killed by an overdose (0.5 ml) of pentobarbital (160 mg ml⁻¹).

The experiments were approved by the ethical committee for animal experimentation of the Medical Faculty, Utrecht University, The Netherlands.

Cardiovascular effects of Arg-Phe containing peptides

Rats were injected i.v. with increasing doses of γ₂-MSH(6–12) (n=6), MERFa (n=7), RFa (n=6), neuropeptide FF (NPFFa; n=6), FMRFa (n=6), acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa; n=6) or desamino-Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa; n=6). The dose range for γ₂-MSH(6–12) was 1.5, 5, 15, 50 and 100 nmol kg⁻¹; for MERFa 25, 50, 100, 200, 300, 400 and 500 nmol kg⁻¹; for acFnLRFa 100, 200, 300, 500, 1000 nmol kg⁻¹ and for RFa, NPFFa, FMRFa and daYFnLRFa 15, 50, 100, 250, 500 and 1250 nmol kg⁻¹.

Effect of C-terminal modifications on cardiovascular actions

Rats were injected i.v. with increasing doses of γ₂-pro¹¹-MSH (n=6), desamino-Tyr-Phe-norLeu-Arg[L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid]-amide (daYFnLR[TIC]a; n=6) or Met-enkephalin (ME; n=8). The dose-range for ME was 25, 50, 100, 200, 300, 400 and 500 nmol kg⁻¹; for γ₂-pro¹¹-MSH and daYFnLR[TIC]a 15, 50, 100, 250, 500 and 1250 nmol kg⁻¹.

Drugs

Phenylephrine was purchased from Sigma Chemical Co., St. Louis, MO, U.S.A.; MERFa, ME, RFa, NPFFa, FMRFa and acFnLRFa from Bachem, Bubendorf, Switzerland; γ₂-MSH(6–12), daYFnLRFa and daYFnLR[TIC]a were synthesized, purified and analysed by mass spectrometry at the National Institute of Public Health and the Environment, Bilthoven, The Netherlands; γ₂-pro¹¹-MSH was kindly donated by Dr R. Adan, Rudolf Magnus Institute for Neurosciences, Utrecht, The Netherlands. All drugs were dissolved in bidestilled water prior to use.

Statistics

The MAP (mmHg) and HR (beats min⁻¹) data are presented as maximal effects (mean changes±s.e.mean) to each dose of injection. This maximal effect is the difference between the maximal response to drug injection and the pre-injection value (measured just before i.v. injection). Over these data a non-linear Hill-fit was performed with the formula: Y=bottom+(top-bottom/1+10^{((logED50−X)*Hillslope)}) in which Y is the MAP or HR response, X is the log of the dose, bottom is the Y value at the bottom plateau; top is the Y value at the top plateau, and LogED₅₀ is the logarithm of the ED₅₀, the concentration that gives a response halfway between bottom and top. The variable Hillslope controls the slope of the curve, which was fitted over the data points. Only those curves were fitted according to this method when a plateau level (E_max, the estimated maximal effect) was reached. The ED₅₀ and E_max reflect a measure of potency and intrinsic activity, respectively.

Baseline MAP and HR (recorded before phenylephrine injection), ED₅₀ and E_max levels were analysed by a one-way Analysis of Variance (ANOVA) and post-hoc Tukey HSD test. A value of P<0.05 was considered significant.

Results

Cardiovascular effects of Arg-Phe containing peptides

Figure 1 shows the MAP and HR response to i.v. injection of γ₂-MSH(6–12), MERFa, RFa, NPFFa, FMRFa, acFnLRFa and daYFnLRFa and the fitted dose-response curve for each peptide. All the tested peptides showed a dose-dependent increase in MAP and HR. The dose-response curves of γ₂-MSH(6–12) was shifted to the left in relation to the other peptides.

Figure 1.

Dose-response relationship for γ₂-MSH(6–12) and various FMRFa-like peptides with respect to their effects on mean arterial blood pressure (MAP) (upper panel) and heart rate (HR) (lower panel) after intravenous administration to conscious, freely moving rats. The results are expressed as absolute maximal change from pre-administration values and as mean±s.e.mean (n=6–7).

Table 2 shows the basal levels of MAP and HR prior to drug administration. Baseline MAP and HR were not significantly different between the 10 groups of rats. ED₅₀ and E_max levels of ΔMAP and ΔHR are shown in Table 2. γ₂-MSH(6–12) showed the most potent cardiovascular effects as compared to the other Arg-Phe containing peptides. daYFnLRFa showed a significant decrease of potency on MAP as compared to FMRFa. The intrinsic activity of MERFa was significantly lower than that of the other Arg-Phe containing peptides.

Table 2.

Basal levels of MAP (mmHg) and HR (beats min⁻¹) prior to phenylephrine administration and ED₅₀ and E_max levels of ΔMAP and ΔHR for γ₂-MSHs and FMRFa-like peptides

Effect of C-terminal modifications on cardiovascular actions

To indicate that the C-terminal Arg-Phe structure is indeed essential for bioactivity, this sequence was modified by substitution of the C-terminal Phe in γ₂-MSH with Pro (γ₂-pro¹¹-MSH), substitution of the C-terminal Phe in daYFnLRFa with L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid (daYFnLR[TIC]a), or by removal of the Arg-Phe-amide structure in MERFa (ME).

Figure 2 shows the MAP and HR response to i.v. injection of γ₂-MSH(6–12), γ₂-pro¹¹-MSH, daYFnLRFa, daYFnLR[TIC]a, MERFa and ME and the fitted dose-response curve for each peptide. The Arg-Phe containing peptides showed a dose-dependent increase in MAP and HR, whereas peptides with a C-terminal modification (γ₂-pro¹¹-MSH, daYFnLR[TIC]a and ME) had no significant effect on the cardiovascular system.

Figure 2.

Dose-response relationship for γ₂-MSH(6–12), γ₂-pro¹¹-MSH, MERFa, ME, daYFnLRFa and daYFnLR[TIC]a with respect to their effects on mean arterial blood pressure (MAP) (upper panel) and heart rate (HR) (lower panel) after intravenous administration to conscious, freely moving rats. The results are expressed as absolute maximal change from pre-administration values and as mean±s.e.mean (n=6–8).

ED₅₀ and E_max levels of ΔMAP and ΔHR for the tested peptides are shown in Table 2. The ED₅₀ levels of the peptides with a C-terminal modification was not measurable. The intrinsic activity (E_max levels) of these peptides was significant lower than that of the Arg-Phe containing analogues.

Discussion

The strong cardiovascular effects of γ₂-MSH(6–12) in conscious rats as described in the present study were consistent with those reported previously by our laboratory (Van Bergen et al., 1995). In that study it was shown that γ₂-MSH(6–12) was more potent than γ₂-MSH, whereas shortening of the C-terminal site of γ₂-MSH resulted in loss of cardiovascular effects, indicating that the bioactivity of γ₂-MSH is carried by the C-terminal site. It has been postulated that the cardiovascular effects of γ₂-MSH in rats are dependent on the C-terminal Arg-Phe sequence (Klein et al., 1985). This agrees with studies of our laboratory and others showing that α-MSH, β-MSH and γ₃-MSH, which do not contain the C-terminal Arg-Phe structure, are devoid of cardiovascular activity in conscious rats (Gruber & Callahan, 1989; Van Bergen et al., 1997b). On the other hand, peptides other than γ-MSHs, which contain the C-terminal Arg-Phe sequence, e.g. the molluscan peptide Phe-Met-Arg-Phe-amide (FMRFa) and mammalian analogues Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide (NPFFa) and Met-enkephalin-Arg-Phe-amide (MERFa) increase MAP and HR in anaesthetized rats (Allard et al., 1995; Barnard & Dockray, 1984a; Mues et al., 1982; Thiemermann et al., 1991; Wong et al., 1985). These studies further suggest that the C-terminal Arg-Phe structure in γ-MSHs is essential for cardiovascular actions. It has to be mentioned that the cardiovascular effects of the FMRFa-like peptides in the latter studies were found in anaesthetized animals. To avoid any effect of anaesthesia on the cardiovascular response, we systemically injected γ₂-MSH(6–12), FMRFa, NPFFa, MERFa and RFa in conscious rats. GTP-γ-S assays and radioligand displacement studies showed that elongation (desamino-Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa)) or modification (acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa)) of Phe at position 1 in FMRFa leads to increased efficacy and potency for the FMRFa receptor in the invertebrate brain (Chin et al., 1994; Heyliger et al., 1998; Payza, 1987). We were interested whether these analogues of FMRFa also showed a stronger effect in vivo and tested these ligands for their cardiovascular action in conscious rats.

Cardiovascular effects of Arg-Phe containing peptides

The results demonstrate that all the tested peptides showed a dose-dependent increase in MAP and HR. Elongation (daYFnLRFa) or modification (acFnLRFa) of the N-terminal site of FMRFa did not lead to a stronger effect on the cardiovascular system. daYFnLRFa even showed a significant decrease of potency on MAP (ED₅₀=2927thinsp;nmol kg⁻¹) as compared to FMRFa (ED₅₀=177 nmol kg⁻¹). These results are in contradiction with those of the above-mentioned binding studies on the FMRFa receptor in invertebrates (Chin et al., 1994; Heyliger et al., 1998; Payza, 1987). On the other hand, our results are supported by radioligand displacement studies by Payza et al. (1993), who indicated that daYFnLRFa shows a decreased efficacy and potency for the NPFFa receptor in rat spinal cord. To our knowledge, there is no literature on the binding and activity capacities of FMRFa-like peptides in the rat brain, which makes it difficult to relate the cardiovascular effects of the present study to specific binding of either the FMRFa or NPFFa receptor. As we found a similar intrinsic activity and potency on the cardiovascular system for FMRFa and NPFFa, we postulate that these peptides share the same receptor. Future binding studies in rat brain tissue are necessary to confirm this hypothesis.

The intrinsic activity of MERFa (E_max=38 mmHg (ΔMAP) and 52 beats min⁻¹ (ΔHR)) was significantly lower than that of the other Arg-Phe containing peptides (E_max=55–65 mmHg (ΔMAP) and 92–124 beats min⁻¹ (ΔHR)). This may be due to the fact that the Met-enkephalin part of MERFa binds on opioid receptors, mediating opposing effects (hypotension and bradycardia) on the cardiovascular system (Douglas & Kitchen, 1992; Hao & Rabkin, 1997), whereas the C-terminal Arg-Phe structure can bind to the FMRFa/NPFFa receptor.

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). It has been shown that α-MSH contains two message sequences for the same biological action (Eberle & Schwyzer, 1976; Schwyzer & Eberle, 1977). Eberle and Schwyzer (1976) reported that the message elements necessary for triggering melanotropic responses are contained in the central tetrapeptide sequence His-Phe-Arg-Trp and C-terminal tripeptide sequence Lys-Pro-Val. Gruber & Callahan (1989) postulated that γ₂-MSH also has dual message sequences: Arg-Trp (at positions 7 and 8) and Arg-Phe (at positions 10 and 11), which are responsible for enhancing cardiovascular activity. This hypothesis agrees with our previous finding that peptides with a single C-terminal Arg-Trp message sequence (γ₂-MSH(1–10), γ₂-MSH(1–8) and fragments of ACTH: ACTH(4–10) and ACTH(4–9)) showed a reduction or even complete loss of cardiovascular action and agrees with the present finding that peptides with a single C-terminal Arg-Phe message sequence (FMRFa, NPFFa, MERFa, RFa, acFnLRFa and daYFnLRFa) showed a reduced potency as compared to γ₂-MSH(6–12), which contains both the Arg-Trp and Arg-Phe structure.

Payza (1987) reported that replacement of the C-terminal Arg-Phe sequence by a Arg-Trp sequence increased the binding potency of FMRFa on the FMRFa receptor in invertebrates. Replacement of the aromatic hydrophobic amino acid by a non-aromatic hydrophobic amino acid (Leu or Pro) decreased the binding potency of FMRFa on the molluscan FMRF receptor (Chin et al., 1994; Payza, 1987). In agreement with findings of Mues et al. (1982), the present study shows that the smallest fragment of FMRFa required for cardiovascular activity was RFa. It is likely that the cardiovascular effects of γ₂-MSH in rats are dependent on the Arg-aromatic hydrophobic amino acid sequence located at or near its C-terminus (Klein et al., 1985), due to binding properties of this structure on the FMRFa/NPFFa receptor.

Relevance of the C-terminal Arg-Phe structure

To indicate that the C-terminal Arg-Phe structure is indeed essential for bioactivity, this sequence was modified by substitution of the C-terminal Phe in γ₂-MSH with Pro (γ₂-pro¹¹-MSH), substitution of the C-terminal Phe in daYFnLRFa with a rigid analogue of Phe, L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid (TIC) (daYFnLR[TIC]a) or by removal of the Arg-Phe structure in MERFa (ME). Systemic injection of these C-terminal modified peptides did not result in significant cardiovascular changes. These results further indicate that cardiovascular effects of γ₂-MSHs and FMRFa-like peptides are dependent on the presence of a C-terminal Arg-Phe sequence.

Inhibition of the baroreceptoreflex

The dose-dependent increase in MAP induced by γ₂-MSH(6–12) is accompanied with a dose-dependent tachycardia. A similar haemodynamic profile is found for the other Arg-Phe containing peptides. This suggests that the baroreceptor reflex-mediated reduction of tonic sympathetic activity due to pressor effects is inhibited by these peptides. This agrees with findings of (Callahan et al. 1985; 1988), who reported that the pressor response to γ₂-MSH in conscious rats was blocked by the ganglionic blocking agent chlorisondamine or by blockade of the catecholamine release from sympathetic nerve terminals with bretylium tosylate. Others showed that pressor responses to Arg-Phe containing peptides in anaesthetized rats was reduced by the ganglionic blocking agent hexamethonium (Barnard & Dockray, 1984b). Furthermore, Thiemermann et al. (1991) showed that systemic injections of FMRFa were associated with an increase in norepinephrine levels, whereas plasma epinephrine levels remained unchanged. These findings indicate that γ₂-MSH and FMRFa-like peptides induce a centrally mediated activation of preganglionic sympathetic efferents. In addition, it is plausible that Arg-Phe containing peptides have direct positive chronotropic action on the heart, as application of FMRFa, MERFa and acFnLRFa on isolated molluscan heart preparations caused cardioacceleration (Painter et al., 1982). However, application of γ-MSH on isolated molluscan (Greenberg et al., 1988) or rat (Van Bergen et al., 1998) heart preparations caused no cardiac effects and in the pithed rat systemically administered γ₂-MSH (Sun et al., 1992; Van Bergen et al., 1998) or FMRFa (Thiemermann et al., 1991) does not exert cardiovascular effects. These findings suggest that γ₂-MSH does not induce cardiovascular action via a peripheral site of action. Further studies have to be performed to understand the exact site and mechanism of cardiovascular actions of Arg-Phe containing peptides.

The results from the present study strongly suggest that γ₂-MSH(6–12), FMRFa and NPFFa share an identical receptor involved in cardiovascular regulation.

Abbreviations

α-MSH

α-melanocyte-stimulating hormone

β-MSH

β-melanocyte-stimulating hormone

γ₂-MSH

γ₂-melanocyte-stimulating hormone

γ₂-MSH(6–12)

Phe-Arg-Trp-Asp-Arg-Phe-Gly

γ₂-pro¹¹-MSH

Lys-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Pro-Gly

ACTH

adrenocorticotropic hormone

acFnLRFa

acetyl-Phe-norLeu-Arg-Phe-amide

daYFnLRFa

desamino-Tyr-Phe-norLeu-Arg-Phe-amide

daYFnLR[TIC]a

desamino-Tyr-Phe-norLeu-Arg-[L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid]-amide

FMRFa

Phe-Met-Arg-Phe-amide

HR

heart rate

MAP

mean arterial pressure

MC

melanocortin

ME

Met-enkephalin

Tyr-Gly-Gly-Phe-Met; MERFa

Met-enkephalin-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide

NPFFa

neuropeptide FF

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide; RFa

Arg-Phe-amide