Abstract
Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC3, MC4, and MC5receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1–24 (agonist at MC4 and MC5 receptors) at a dose of 160 μg/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of γ1-melanocyte stimulating hormone (selective agonist at MC3 receptors) was completely ineffective. The selective antagonist at MC4 receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1–24, with the effect being complete either at the i.v. dose of 200 μg/kg or at the i.c.v. dose of 5 μg/rat (17–20 μg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC4receptors in the brain.
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 γ1- and γ2-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 γ3-MSH, α-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), α-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-α (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-α (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 seeBertolini, 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 ω-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 α-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
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).
Volume-controlled hemorrhagic shock was induced by stepwise bleeding from the venous catheter over a period of 25 to 30 min until mean arterial pressure (MAP), automatically calculated by the polygraph, reduced to, and stabilized at, 22 to 25 mm Hg. The total bleeding volume was 2.26 ± 0.16 ml/100 g b.wt. [overall mean ± S.E. from all rats subjected to bleeding (n = 155); the volume was similar for each experimental group, ranging from 2.18 ± 0.22 to 2.30 ± 0.19, P > .05, ANOVA].
Drugs and Treatments.
ACTH-(1–24) (Sigma Chemical Co., St. Louis, MO) was chosen as agonist at MC4 (maximum potency and efficacy equal to α-MSH and β-MSH) (Gantz et al., 1993b) and MC5 receptors (maximum potency and efficacy equal to α-MSH and [Nle4,d-Phe7]α-MSH) (for review, see Hol et al., 1995). γ1-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
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 γ1-MSH, also injected 5 min after the termination of bleeding, was completely ineffective (Fig.2).
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).
Discussion
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, γ1-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 α- and β-MSH) (Gantz et al., 1993b) and MC5 receptors (equal to α-MSH and [Nle4,d-Phe7]α-MSH) (Griffon et al., 1994). The MC3 receptors have the highest affinity for γ1-MSH and desacetyl-α-MSH (Schiöth et al., 1995), and γ-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, MC3, 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 (γ1-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.
Acknowledgments
We thank Dr. Felikss Mutulis for synthesis of HS014.
Footnotes
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Send reprint requests to: Salvatore Guarini, Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, via G.Campi 287, 41100 Modena, Italy. E-mail:Guarini.Salvatore{at}unimo.it
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↵1 This work was supported in part by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica and Consiglio Nazionale delle Ricerche, Italy, and the Swedish MRC (04X-05957).
- Abbreviations:
- ACTH
- adrenocorticotropin
- MSH
- melanocyte-stimulating hormone
- HR
- heart rate
- PP
- pulse pressure
- RR
- respiratory rate
- MAP
- mean arterial pressure
- Received June 14, 1999.
- Accepted August 3, 1999.
- The American Society for Pharmacology and Experimental Therapeutics