A carboxy-terminal peptide of the alpha 1-subunit of the dihydropyridine receptor inhibits Ca(2+)-release channels.

Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor). Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction. Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins. A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both [3H]ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers. A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide. These peptides slow the rate at which [3H]ryanodine binds to its high-affinity binding site and decrease the rate at which [3H]ryanodine dissociates from this site. A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects. This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.

Interaction between ryanodine and neomycin binding sites on Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.

Loss of adenosine-induced negative inotropic effect in hyperexcited rabbit hearts: relationship to PKC.

Adenosine produced a negative inotropic effect in hearts isolated from calm rabbits but not from those exhibiting alarm behavior during handling. This study was conducted to determine whether protein kinase C (PKC) activation is responsible for the loss of adenosine-induced negative inotropism in the hearts of hyperexcited rabbits. Adenosine (10 microM) decreased myocardial contractility (dP/dtmax) in the hearts of calm, but not hyperexcited, rabbits but decreased heart rate (HR) and coronary perfusion pressure (PP) in the hearts of both calm and hyperexcited animals. During infusion of calphostin C (200 nM), a PKC inhibitor, adenosine also decreased dP/dtmax in the hearts of hyperexcited rabbits. Calphostin C did not alter the actions of adenosine in the hearts of calm rabbits. Agents that stimulate PKC directly [phorbol 12,13-dibutyrate (PDBu), 1 nM] or indirectly [norepinephrine (NE), 3 nM; angiotensin II (ANG II), 5 nM] abolished the adenosine-induced decrease in dP/dtmax but not HR or PP in the hearts of calm rabbits. During calphostin C, infusion of PDBu, NE, and ANG II failed to prevent the adenosine-induced decrease in dP/dtmax. These data suggest that the lack of a negative inotropic effect of adenosine in hyperexcited rabbits is due to an increase in PKC activity.

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Naloxone-induced potentiation of cardiac alpha-2 adrenoceptor-mediated depression of neurogenic tachycardia.

The objective of this investigation was to test the hypothesis that naloxone directly activates alpha-2 adrenoceptors to cause depression of neurogenic tachycardia as suggested in an earlier investigation (Naloxone-Induced Bradycardia in Pithed Rats: Evidence for an Interaction with the Peripheral Sympathetic Nervous System and Alpha-2 Adrenoceptors. J. Pharmacol. Exp. Ther. 296: 916-926, 1992). Bolus doses of naloxone in a range of 10-1000 micrograms/kg i.v., administered in the presence of sustained neurogenic tachycardia (108 +/- 10 beats per min), resulted in a dose-dependent inhibition of neurogenic tachycardia with a maximum inhibitory response of 21% and an ED50 of 55 +/- 2.3 micrograms/kg. The inhibition of the naloxone-induced inhibition of neurogenic tachycardia was antagonized by phentolamine (5 mg/kg i.v.) and rauwolscine (0.5 mg/kg i.v.), but not prazosin (0.1 mg/kg i.v.). In the absence of sympathetic nerve activity, low doses of naloxone (10-300 micrograms/kg i.v.) had no effect on heart rate. These data suggest that naloxone in lower doses (10-1000 micrograms/kg i.v.) is a partial agonist at prejunctional alpha-2 adrenoceptors. In the presence of a steady-state maximum response (21% inhibition of neurogenic tachycardia) caused by naloxone infusion (100 and 1000 micrograms/kg/min i.v.), the ED50 of the preferential alpha-2 adrenoceptor agonist, UK14304-18, was not shifted to the right, but instead shifted to the left. This suggests that naloxone-induced depression of the neurogenic tachycardia does not involve the direct activation of alpha-2 adrenoceptors, but involves the potentiation of alpha-2 adrenoceptor-mediated inhibition of heart rate through an unknown mechanism.

Involvement of inhibitory dopamine-2 receptors in resting bradycardia in exercise-conditioned rats.

The purpose of this investigation was to examine the underlying cause for the resting bradycardia and lower resting blood pressure demonstrated in conscious rats that performed 12 wk of treadmill exercise conditioning. The influence of inhibitory dopamine (DA2) receptors and alpha 2-adrenoceptors, which are known to mediate bradycardia and hypotension, was assessed in exercise-conditioned (EC) and nonexercised conditioned (NC) rats. To accomplish this, preferential DA2 and alpha 2-agonists and antagonists were administered at rest to conscious rats after they participated in an exercise conditioning program. The results obtained with the DA2 antagonist metoclopramide (15 mg/kg ip) alone suggest that there is physiological activation of cardiovascular DA2 receptors in EC rats but not in NC rats. Furthermore, the results obtained with the DA2 agonist bromocriptine (1.5 mg/kg ip) suggest that the DA2 receptor-mediated bradycardia and hypotension are greater in EC rats than in NC rats. In addition, heart rate and blood pressure responses to the alpha 2-agonist clonidine (0.1 mg/kg ip) and antagonist yohimbine (1 mg/kg ip) were not different between EC and NC rats. These data suggest that enhanced DA2 receptor influence accounts, in part, for the resting bradycardia and lower resting blood pressure demonstrated in EC rats after 12 wk of exercise conditioning.

Naloxone-induced bradycardia in pithed rats: evidence for an interaction with the peripheral sympathetic nervous system and alpha-2 adrenoceptors.

Earlier experiments performed in this laboratory have demonstrated that naloxone infusion (1 mg/kg/min i.v.) into conscious rats results in a bradycardia that has a peripheral component, is dependent on a certain level of sympathetic activity and is sensitive to alpha adrenoceptor blockade (5 mg/kg of phentolamine i.v.). The main objective of this investigation was to examine the underlying mechanism(s) responsible for the peripherally mediated naloxone-induced bradycardia, and to test the hypothesis that naloxone interacts with peripheral inhibitory alpha adrenoceptors associated with depression of peripheral sympathetic activity. Naloxone infusion (1 mg/kg/min i.v.) in pithed rats, in the absence of sympathetic nerve activation, resulted in a bradycardia that could not be blocked by 1 mg/kg (i.v.) of atropine, 5 mg/kg (i.v.) of phentolamine, 0.1 mg/kg (i.v.) of prazosin or 0.5 mg/kg (i.v.) of rauwolscine. Isoproterenol or norepinephrine-induced tachycardia was not blocked by naloxone infusion, suggesting that naloxone does not antagonize the postjunctional activation of cardiac adrenoceptors to cause bradycardia. In the presence of sympathetic nerve activity, naloxone depresses neurogenic tachycardia. This effect was blocked completely by 5 mg/kg (i.v.) of phentolamine or 0.5 mg/kg (i.v.) of rauwolscine, but not 0.1 mg/kg (i.v.) of prazosin or 1 mg/kg (i.v.) of atropine. The results of this investigation suggest that the naloxone-induced bradycardia in pithed rats is mediated postjunctionally and prejunctionally, and that this prejunctional effect is dependent on sympathetic nerve activity and inhibitory alpha-2 adrenoceptors. Furthermore, these results confirm results obtained from conscious rats in an earlier investigation.

A method for maintaining and protecting chronic arterial and venous catheters in conscious rats.

The ability to monitor arterial blood pressure and heart rate directly, as well as to sample venous blood, or inject pharmaceutical agents intravenously is important in pharmacological studies of the cardiovascular system. The rat is a frequently used and accepted animal model for cardiovascular investigations, especially those relating to hypertension. Even though the rat is a major model for these studies, the size of the rat has made it difficult to maintain catheters for a long period of time. Although there have been previous methods available, the authors report on an improved method to implant, maintain, and protect arterial and venous catheters in conscious rats for extended periods of time. A Silastic/Tygon catheter is implanted intraarterially and intravenously, exteriorized, and protected with a spring device. Catheters remained patent throughout a 5-week period during which time direct blood pressure recordings were obtained and baroreflexes were evaluated in conscious, unrestrained rats. The described design and methods provide an inexpensive means to maintain chronically implanted venous and arterial catheters in the conscious rat. Furthermore, rats may be gang housed.