Introduction

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Melanocortin peptides have important cardiovascular effects. In conscious rats, as well as in rats under light urethane anesthesia (where reflexes and sufficient sympathetic tone are maintained), the adrenocorticotropin fragment 4-10 [ACTH-(4-10)] and ₁- and ₂-melanocyte- stimulating hormones [MSH; 10 times more potent than ACTH-(4-10)] induce a dose-dependent, short-lasting increase in blood pressure, heart rate (HR), and pulse amplitude following their i.v. administration (for review see Gruber and Callahan, 1989; Versteeg et al., 1998). However, melanocortin peptides with a longer C-terminal extension, including ₃-MSH, -MSH, ACTH-(1-17), ACTH-(1-24), etc., are devoid of these cardiovascular effects in the normotensive, normovolemic animal (Klein et al., 1985; Bertolini et al., 1986c, 1989). However, melanocortin peptides lacking the C-terminal Arg-Phe sequence [ACTH-(4-10), -MSH, ACTH-(1-17), ACTH-(1-24), etc.] have dramatic cardiovascular effects in severe hypotensive conditions (for review see Bertolini, 1995).

In a model of volume-controlled hemorrhagic shock in rats and dogs that caused the death of all saline-treated animals within 20 to 30 min, the i.v. bolus injection of any of these peptides induces, within a few minutes, an adrenal-independent, dose-dependent (minimum and maximum ED 20 and 160 µg/kg, respectively) restoration of cardiac output, total peripheral vascular resistance index, arterial pressure, pulse amplitude, and tissue blood flow, with gradual normalization of arterial and venous pH and base excess, as well as venous tension of O₂ (PO₂) and CO₂ (PCO₂), venous oxygen saturation (SO₂), and lactate (Bertolini et al., 1986a,b,c; 1989; Bazzani et al., 1992). Moreover, there is a highly significant reduction of free radicals (Guarini et al., 1996), nitric oxide (Guarini et al., 1997), and tumor necrosis factor- (Altavilla et al., 1998) blood levels. The survival time is greatly increased: 44 ± 18 h (range 15 to 312 h; n = 18; mean survival time in saline-treated animals 26 ± 1 min; n = 20) (Bertolini et al., 1989). The temporary reversal of hemorrhagic shock induced by these peptides is associated with a large increase in the volume of circulating blood (Guarini et al., 1987a; Bertolini et al., 1989), as the consequence of the mobilization of the peripherally pooled residual blood. Indeed, the antishock effect of these peptides is impaired in animals deprived of blood reservoirs (splenectomized animals and animals subjected to ligature of the suprahepatic veins) (Guarini et al., 1987a, 1988; Bertolini et al., 1989). The restoration of the blood flow in vital organs greatly extends the time limit for an effective and curing blood reinfusion. Although all rats reinfused with their own shed blood at 15 min after hemorrhage die within 6.6 ± 4.4 h, a substantial number of rats treated with a melanocortin peptide shortly after bleeding (within 5 min) survive, even if blood reinfusion is performed 30, 60, or 120 min later (Bertolini et al., 1989). This resuscitating effect of melanocortins also has been confirmed in a model of hypovolemic shock produced in rabbits by the graded occlusion of the inferior vena cava (Ludbrook and Ventura, 1995) and in the splanchnic artery occlusion shock in rats (Squadrito et al., 1999), as well as in human subjects with hemorrhagic or cardiogenic shock (Bertolini et al., 1987; Pinelli et al., 1989; Noera et al., 1989, 1991).

The studies on the mechanisms underlying the antishock effect of melanocortins suggest that in conditions of failure of the circulatory homeostasis, these peptides inhibit the overproduction of tumor necrosis factor- (Altavilla et al., 1998) and nitric oxide (Guarini et al., 1997) (these effects are probably related), and activate or restore a complex vasomotor reflex that eventually leads to the mobilization of the peripherally pooled residual blood (for review see Bertolini, 1995), and which is seemingly obtunded by the massive release of endogenous opioids that occurs in such conditions (Bernton et al., 1985; Schadt, 1989). Opioids inhibit sympathetic outflow and noradrenaline release from sympathetic terminals (Schadt, 1989), whereas melanocortins have an opposite effect (Szabo et al., 1987).

Together, these effects of melanocortins would remove the main causes of hemorrhage-induced circulatory decompensation, namely, the blunted release of noradrenaline from sympathetic terminals and the reduced responsivity of resistance vessels to noradrenaline. The vasomotor reflex that melanocortins activate/restore in shock conditions (Bertolini, 1995) includes an essential brain loop. Indeed, the shock reversal induced by the i.v. injection of melanocortins is prevented or greatly impaired by 1) bilateral vagotomy at the cervical level (Guarini et al., 1986); 2) the i.v. injection of capsaicin (Guarini et al., 1992) (which induces a defunctionalization of primary afferent-Substance P containing-nerve fibers) or of a Substance P antagonist (Guarini et al., 1992); 3) the i.c.v. injection of hemicholinium-3 (Guarini et al., 1990a); 4) the blockade of brain M₃-muscarinic receptors (Guarini et al., 1990b); and 5) the i.c.v. injection of the N-calcium channel blocker -conotoxin (Guarini et al., 1993). Finally, shock reversal also can be obtained with the i.c.v. injection of melanocortins (Guarini et al., 1987b).

Molecular cloning of five melanocortin receptor subtypes (MC₁-MC₅) (Chhajlani and Wikberg, 1992; Mountjoy et al., 1992; Gantz et al., 1993a,b; Chhajlani et al., 1993; Schiöth et al., 1996; Adan and Gispen, 1997) has provided tools for the study of the mechanisms of the effect of melanocortins. MC₁ is the specific -MSH receptor expressed in melanocytes, melanoma cells, and macrophages; MC₂ is the ACTH receptor expressed in the adrenal gland; and MC₃, MC₄, and MC₅ receptors are also (MC₅), mainly (MC₃) or exclusively (MC₄) expressed in brain tissue. The present study was aimed at defining whether (and, in the affirmative, which) brain melanocortin receptors are involved in the antishock effect of melanocortin peptides.

	Materials and Methods

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Animals and Surgery. Wistar rats of both sexes (Charles River Breeding Laboratories, Calco, Como, Italy), weighing 250 to 280 g, were used. They were housed four per cage, males and females separately, with food in pellets (TRM, Harlan; Teklad Premier Laboratory Diet, Madison, WI) and tap water freely available, under the temperature (22 ± 1°C), humidity (60%), and ventilation conditions advised by the European Community ethical regulations on the care of animals for scientific research, on a 12 h light/dark cycle (light phase: 0700-1900 h). The animals were acclimatized to our housing conditions for at least 1 week before being used. A group of rats was prepared for i.c.v. treatment by stereotaxically implanting stainless steel guide cannulas (23 gauge; Plastic Products Co., Roanoke, VA) into a brain lateral ventricle (Paxinos and Watson, 1982), under ketamine plus xylazine anesthesia [115 + 2 mg/kg i.p.; Farmaceutici Gellini, Aprilia, Italy and Bayer, Milano, Italy, respectively], and by fixing them to the skull with screws and dental acrylic cement. A removable plug, which extended 0.5 mm below the tip of the guide cannula was kept in place until drug injection. Correct placement was verified at the end of the experiment by injecting 4 µl of toluidine blue dye, followed by decapitation under ethyl ether anesthesia and dissection of the brain. Data obtained from incorrectly implanted rats (5 of 65) were discarded. The experiments were performed under urethane anesthesia (1.25 g/kg i.p.). Urethane (Fluka AG, Buchs, Switzerland) was chosen because it provides long-lasting and stable general anesthesia with minimal interference with cardiovascular regulatory functions. After heparinization (heparin sodium; 600 I.U./kg i.v.), rats were instrumented with indwelling polyethylene catheters in a common carotid artery and an iliac vein. Systemic arterial pressure and pulse pressure (PP) were recorded by means of a pressure transducer (P23 Db; Statham, Oxnard, CA) coupled to a polygraph (Battaglia-Rangoni, Bologna, Italy). HR was automatically calculated from the pulse wave by the same polygraph. Respiratory rate (RR) was recorded by means of three electrodes s.c. implanted on the chest and connected to the polygraph through an ARI A380 preamplifier (Battaglia-Rangoni).

Drugs and Treatments. ACTH-(1-24) (Sigma Chemical Co., St. Louis, MO) was chosen as agonist at MC₄ (maximum potency and efficacy equal to -MSH and -MSH) (Gantz et al., 1993b) and MC₅ receptors (maximum potency and efficacy equal to -MSH and [Nle⁴, D-Phe⁷]-MSH) (for review, see Hol et al., 1995). ₁-MSH (Bachem, Bubendorf, Switzerland) was chosen as selective agonist at MC₃ receptors (for review, see Hol et al., 1995; Schiöth et al., 1995; Versteeg et al., 1998). The cyclic MSH analog HS014, synthesized with solid phase approach and purified by HPLC, was chosen as highly potent and selective antagonist at MC₄ receptors (Schiöth et al., 1998). The correct molecular weight of the peptide was confirmed by mass spectrometry. All these peptides were freshly dissolved in saline shortly before use. The i.v. injections were in a volume of 1 ml/kg; the i.c.v. injections were in a volume of 5 µl/rat. Control animals received equivolume amounts of saline.

Statistics. MAP, PP, HR, and RR values, and total bleeding volumes were analyzed by means of ANOVA followed by Student-Newman-Keuls test. Survival rates were analyzed by Fisher's exact probability test.

Animal Ethics. Experimental procedures were carried out in accordance with guidelines of the European Community, local laws and policies (D.L.vo 116/92).

	Results

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The baseline values of the recorded parameters (MAP, PP, HR, RR) were not significantly different in any of the experimental groups. As repeatedly described (for review see Bertolini, 1995), the acute and severe hypovolemia induced in our model of volume-controlled hemorrhagic shock in anesthetized rats was incompatible with survival and, hence, all saline-treated animals died within 30 min after saline injection (Figs. 1, 2, and 4). The i.v. bolus injection of ACTH-(1-24) 5 min after the termination of bleeding, at the maximum ED (Bertolini et al., 1989) of 160 µg/kg, produced, within a few minutes, an almost complete restoration of cardiovascular and respiratory functions that 10 to 15 min after treatment were not significantly different from baseline (Fig. 1). This effect of ACTH-(1-24) remained unchanged throughout the observation period (60 min). An i.v. equimolar dose (54 nmol/kg) of ₁-MSH, also injected 5 min after the termination of bleeding, was completely ineffective (Fig. 2).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Influence of i.v. pretreatment with the MC4 antagonist HS014 (25-200 µg/kg) on the resuscitating effect of ACTH-(1-24) (160 µg/kg i.v.) in rats subjected to hemorrhagic shock. A, MAP; B, PP; C, HR; D, RR. Values are means ± S.E., n = 6 to 8 rats per group. *P < .05, at least, versus the corresponding value in saline-pretreated and ACTH-treated rats (Student-Newmann-Keuls test). In some cases, for the sake of clarity, S.E.M. was omitted. Percentage of surviving animals 60 min after treatment (value at the end of lines): **P < .005 versus the saline-ACTH group (Fisher's test).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of i.v. treatment with 1-MSH or HS014, at doses (81 and 91 µg/kg, respectively) equimolar to 160 µg/kg ACTH-(1-24), in rats subjected to hemorrhagic shock. Only data concerning MAP are shown; the effect on PP, HR, and RR was similar. Values are means ± S.E., n = 6 to 8 rats per group. *P < .05, at least, versus the corresponding value in saline-treated rats (Student-Newmann-Keuls test). In some cases, for the sake of clarity, S.E.M. was omitted. Percentage of animals surviving 60 min after treatment (value at the end of lines): **P < .005 versus saline group (Fisher's test).

HS014, although having no influence per se on cardiovascular and respiratory functions, both in normal, nonbled rats (Fig. 3) and in hemorrhage-shocked rats (Fig. 2), dose-dependently prevented the antishock effect of ACTH-(1-24) that was completely antagonized by HS014 either at the i.v. dose of 200 µg/kg (Fig. 1) or at the i.c.v. dose of 5 µg/rat (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Influence of i.v. (100 and 200 µg/kg) or i.c.v. (5 µg/rat) treatment with HS014 on cardiovascular and respiratory functions in normal, nonbled rats. Saline, 1 ml/kg i.v. or 5 µl/rat i.c.v. Only data concerning MAP are shown; the effect on PP, HR, and RR was similar. Values are means ± S.E., n = 6 to 8 rats per group. In some cases, for the sake of clarity, S.E.M. was omitted. Statistical assessment between groups was not significantly different (ANOVA).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Influence of i.c.v. pretreatment with the MC4 antagonist HS014 (0.5-5 µg/rat) on the resuscitating effect of ACTH-(1-24) (160 µg/kg i.v.) in rats subjected to hemorrhagic shock. Only data concerning MAP are shown; the effect on PP, HR, and RR was similar. Values are means ± S.E., n = 6 to 8 rats per group. *P < .05, at least, versus the corresponding value in saline-pretreated and ACTH-treated rats (Student-Newmann-Keuls test). In some cases, for the sake of clarity, S.E.M. was omitted. Percentage of animals surviving 60 min after treatment (value at the end of lines): **P < .05 and ***P < .005 versus the saline-ACTH group (Fisher's test).

	Discussion

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Our present results confirm that in a condition of severe hemorrhagic shock invariably causing the death of all saline-treated animals within 30 min, the i.v. bolus injection of ACTH-(1-24) induces within a few minutes an almost complete and steady normalization of cardiovascular and respiratory parameters. Furthermore, these results show that a selective antagonist at MC₄ receptors (HS014) (Schiöth et al., 1998), either i.v.- or i.c.v.-injected, dose-dependently prevents the shock reversal induced by ACTH-(1-24); the antagonism being complete after an i.c.v. dose of 5 µg/rat or an i.v. dose of 200 µg/kg. Moreover, ₁-MSH, a selective agonist at MC₃ receptors, injected in an i.v. bolus at a dose equimolar to the maximum ED of ACTH-(1-24) was completely ineffective in our experimental condition of hemorrhagic shock.

The MC₃, MC₄, and MC₅ receptors are expressed in the brain (for review, see Hol et al., 1995; Adan and Gispen, 1997). ACTH-(1-24) has maximum agonist potency and efficacy at MC₄ (equal to - and -MSH) (Gantz et al., 1993b) and MC₅ receptors (equal to -MSH and [Nle⁴, D-Phe⁷]-MSH) (Griffon et al., 1994). The MC₃ receptors have the highest affinity for ₁-MSH and desacetyl--MSH (Schiöth et al., 1995), and -MSHs are selective agonists at MC₃ receptors (for review, see Versteeg et al., 1998). HS014 is a cyclic MSH analog that is a potent and selective antagonist at MC₄ receptors and a partial agonist at MC₁ and MC₅ receptors; its selectivity for the MC₄ receptors is 34-, 17-, and 220-fold higher than that for the MC₁, MC_3, and MC₅ receptors, respectively (Schiöth et al., 1998).

Our present data show that reversal of hemorrhagic shock is produced by the selective agonist at MC₄ and MC₅ receptors [ACTH-(1-24)], but not by a selective agonist at MC₃ receptors (₁-MSH), and that the antishock effect of ACTH-(1-24) can be completely prevented by a selective antagonist at MC₄ receptors (HS014), injected either i.v. or i.c.v.; the i.c.v. ED being 10 to 15 times lower than the i.v. ED.

Thus, the present results further confirm that the mechanism of action of melanocortins in hemorrhagic shock reversal includes the involvement of a brain pathway. Moreover, these data strongly suggest that it is the MC₄ receptor that mediates the hemorrhagic shock reversal caused by melanocortin peptides. This may suggest that selective MC₄ receptor agonists would be specifically effective in shock conditions.