Motilin effects on the heart and blood vessels of the pithed rat

Motilin, a 22 amino acid polypeptide was shown to affect smooth muscle tone in the gastrointestinal tract. However, its widespread distribution in peripheral and central components of the autonomic nervous system suggest a role in other functions such as regulation of vascular tone and hemodynamic variables. Therefore, the effect of motilin on vascular tone, cardiac rhythm and blood vessel response to pressor stimuli was studied in the pithed rat. It is shown that motilin produces a prolonged depressor effect. The depressor responses were dose dependent at the range of 30 - 300 nmol/kg (max. decrease: -22 +/- 4 mmHg). In addition, motilin attenuated pressor responses to vasopressin, leukotriene D4, and the pressor effect evoked by complete spinal cord stimulation. Motilin did not affect the basal heart rate nor did it alter sympathetically induced heart rate acceleration. Motilin did not affect the circulatory level of norepinephrine or epinephrine at resting state or of norepinephrine released by spinal cord stimulation; motilin significantly suppressed epinephrine released by spinal cord stimulation. These data suggest a role for motilin in regulation of blood vessel tone by direct action on the vascular smooth muscle. In addition, motilin might play a role in regulation of epinephrine release from the adrenal medulla.     

The effect of μ, δ, and ε opioid receptor agonists on heart rate and blood pressure of the pithed rat

Opioid peptides and opioid receptors are found in the hearts of various species. Opioid peptides were also shown to modulate norepinephrine inducing changes in atrial rate, in vitro. Since we have recently shown a predominance of kappa and delta receptors in the rat atria, we found it of interest to study the role of highly selective opioid agonists on spontaneous and sympathetically stimulated heart rate. The pithed, artificially ventillated rat was used in these studies. D-Ala2-D-Leu5-enkephalin (DADL), was used as an delta-agonist, D-Ala2-MePhe4-Gly-ol5-enkephalin (DAGO) as a highly selective mu-agonist; Dynorphin A (1-17) as a kappa-agonist and beta-endorphin (beta-END) as a mixed epsilon-delta-mu agonist. Naloxone was used as an opiate antagonist. None of the above opioid peptides changed the basal blood pressure and heart rate at 1-100 nmol/kg except Dyn A-(1-17) which produced a brief depressor response (-15 +/- 2 mmHg, p less than 0.01). Stimulation of the spinal cord (50 v, 1 msec, 1 Hz, 30 sec) produced consistant pressor and cardiac accelerating responses. None of the opioid peptides studied blocked or enhanced the increase in blood pressure or heart rate produced by spinal cord stimulation. The depressor effect of the high dose of Dyn A-(1-17) was not blocked by naloxone. These results suggest that mu, delta or kappa opioid receptors in the rat heart have no role in the regulation of basal or sympathetically driven heart rate. Our data also suggest no role for these opioid receptors in modulation of basal arterial tone or norepinephrine-induced arteriolar constriction.     

Plasma and organ catecholamine levels following stimulation of the rat insular cortex.

The posterior insular cortex of the rat contains an area of cardiac chronotropic representation within which tachycardia sites occur rostrally to those producing bradycardia. In the current study using ketamine-anesthetized rats, the insular cortex was stimulated for 1 h using a phasic technique synchronized with the cardiac cycle. Tachycardia was associated with an increase in plasma norepinephrine concentration; epinephrine remained unchanged. This indicates a neural origin of the norepinephrine increment. The tachycardia response was completely blocked by atenolol. Plasma catecholamine levels remained unchanged during stimulation of insular bradycardia sites. Atenolol was without effect during stimulation-induced bradycardia which was completely blocked by atropine. Total cardiac norepinephrine concentration inversely correlated with change in heart rate during stimulation of tachycardia sites. No correlation between intracardiac catecholamines and heart rate variables was found for the bradycardia or control sites. These results indicate that in the ketamine-anesthetized rat, whereas insular stimulation-induced tachycardia is dependent on the sympathetic nervous system, bradycardia elicited by insular cortex stimulation is mediated by parasympathetic mechanisms. No correlation was identified between renal or skeletal muscle norepinephrine levels and any heart rate parameter. This implies that the sympathetic effects of phasic insular microstimulation may be exerted mainly on cardiac nerves, and less so in other visceral beds.     

Hypothalamic sites for cardiovascular and sympathetic modulation by prostaglandin E2

Prostaglandin E₂ (PGE₂, 0.5–5 nmol/kg) injected into the lateral cerebral ventricle of the rat increased the systemic blood pressure and heart rate in a dose dependent manner. These effects were accompanied by increases in plasma norepinephrine and epinephrine concentration. Injection of the low dose of prostaglandin E₂ into discrete hypothalamic nuclei induced a marked increase in heart rate, a moderate increase in the arterial blood pressure and a significant elevation of plasma norepinephrine level. This study suggests a possible central role for PGE₂ in modulation of cardiovascular dynamics and sympathetic nervous activity.     

Autonomic nerve and cardiovascular responses to changing blood oxygen and carbon dioxide levels in the rat

Contribution of autonomic nervous system activity to the heart rate and blood pressure responses during chemoreceptor excitations by systemic hypoxia and hypercapnia and to hyperoxia and hypocapnia was analyzed in the urethane-anesthetized, artificially ventilated rats. Systemic hypoxia induced a co-activation of two antagonistic nerves: an increase in cardiac sympathetic and in cardiac vagal efferent nerve discharges. Increased heart rate was due to predominance of the cardiac sympathetic over the cardiac vagal activation. In spite of a marked reflex increase in the renal and cardiac sympathetic nerve activities, the local vasodilator effect of hypoxia prevented consistent changes in arterial blood pressure. Bilateral section of the carotid sinus nerves (CSN) mostly abolished autonomic nerve responses and produced a profound decreases in the blood pressure during hypoxia. Hyperoxia elicited a pressor response due to peripheral vasoconstriction with no significant change in the autonomic nerve activities except for a decrease in the cardiac sympathetic nerve discharges. Hypercapnia significantly increased blood pressure and renal nerve sympathetic activity. In contrast to hypoxia, hypercapnia excited cardiac sympathetic and inhibited cardiac vagal activity. This reciprocal effect did not elicit neurogenic cardioacceleration, because it was masked by the local inhibitory action of CO2 on the heart rate. The increase in sympathetic activities and in blood pressure during hypercapnia persisted after bilateral CSN section indicating that the responses were mediated by central rather than by peripheral chemoreceptors. Hypocapnia produced a significant increase in cardiac vagal discharges yet a cardioacceleratory response occurred due to the local effect upon heart rate. The present results indicate that in the rat, autonomic nervous responses differ depending on the type, i.e. hypoxic or hypercapnic, chemoreceptor stimuli. Reflex heart rate and blood pressure responses do not follow the autonomic nerve activities exactly. Circulatory responses are greatly modified by local peripheral effects of hypoxic, hyperoxic, hypocapnic or CO2 stimuli on the cardiovascular system. Species differences characterizing the autonomic nerve responsiveness to chemical stimuli in the rat are described.     

Cardiovascular responses to electrical stimulation of sympathetic nerves in the pithed mouse

The pithed rat model has been used extensively to study peripheral cardiovascular responses to electrical stimulation of the sympathetic nervous system, as pithing eliminates central and reflex effects. However, since the transgenic mouse has become a standard and economical model organism, an electrically stimulated pithed mouse would facilitate a variety of studies. We have developed surgical techniques, drug doses and stimulation parameters for an electrically stimulated pithed mouse to study peripheral sympathetic nerve effects on blood pressure. Similar to the pithed rat, the pithed mouse showed voltage and frequency-dependent blood pressure responses to a pulsed train of electrical stimuli. In addition, alpha-adrenergic stimulation with phenylephrine gave a marked systolic pressor response, while the beta2 agonist salbutamol lowered diastolic blood pressure. Furthermore, pithed transgenic mice unable to synthesize catecholamines in adrenergic cells displayed smaller pressor responses than pithed control mice. In summary, the electrically stimulated pithed mouse can be used to study peripheral effects of the sympathetic system on cardiovascular dynamics unencumbered by central responses.     

Beta-endorphin-induced increases in plasma epinephrine, norepinephrine and dopamine in rats: inhibition of adrenomedullary response by intracerebral somatostatin

Synthetic human beta-endorphin, 7.25 nmol intracisternally, in unanesthetized, freely moving, chronically cannulated, adult male rats increased plasma concentrations of all 3 catecholamines: epinephrine, norepinephrine and dopamine, for the 2 h period studied. Blockade of these endorphin effects by the prior systemic administration of naloxone supports mediation of the effects at opioid receptors. Acute systemic administration of guanethidine, which decreases norepinephrine release induced by sympathetic nerve stimulation, blunted the plasma norepinephrine response to intracerebral beta-endorphin. Thus, it seems likely that in addition to secretion by adrenal medulla a considerable portion of the beta-endorphin-induced increase in norepinephrine is derived from sympathetic nerve endings. Simultaneous intracisternal administration of another neuropeptide, somatostatin, together with beta-endorphin markedly inhibited the plasma epinephrine response to beta-endorphin, while decreasing the dopamine and norepinephrine responses to a much lesser degree. The dats suggest that beta-endorphin stimulates central sympathetic outflow to both adrenal medulla and sympathetic nerve endings, and further that somatostatin inhibits the effect of endorphin to stimulate outflow to adrenal medulla but does not affect outflow to sympathetic nerve endings.     

Facilitation of ACTH secretion by morphine is mediated by activation of CRF releasing neurons and sympathetic neuronal pathways

Exogenously applied opioid agonists have a stimulatory effect on adrenocorticotropic hormone (ACTH) secretion. The present experiments were designed to examine the mechanisms involved in the stimulatory effect of the mu-receptor agonist morphine on ACTH release in chronically cannulated, freely moving, non-stressed rats. Morphine (7.5 mg/kg, i.v.) treatment was followed by a significant increase in plasma levels of ACTH. Pretreatment with the peripheral ganglionic blocker chlorisondamine (3 mg/kg, i.p.) attenuated the response to morphine. The morphine stimulatory effect was also partially inhibited if the rats were pretreated with a specific antiserum to corticotropin-releasing factor (CRF). In rats given both CRF antiserum and chlorisondamine, the plasma ACTH levels remained unchanged after morphine application. These findings indicate that morphine stimulates the release of ACTH by activating both CRF-secretion and peripheral sympathetic neuronal pathways.     

Neonatal methylmercury poisoning in the rat: effects on development of peripheral sympathetic nervous system. Neuronal participation in methylmercury-induced cardiac and renal overgrowth

The effects of neonatal CH3-Hg exposure on development and function of peripheral catecholaminergic synapses were examined by measuring tissue norepinephrine (NE) levels and turnover rates and cardiac biochemical responses to sympathetic reflex stimulation. In the rat, cardiac sympathetic neurotransmission normally develops towards the end of the first week postnatally; however, pups given CH3-Hg showed responses to sympathetic reflex stimulation as early as 2 days of age. The accelerated maturation of cardiac sympathetic effect was accompanied by initial enhancement of NE levels and turnover. This effect appeared to be specific to the heart, as kidney displayed subnormal NE levels in CH3-Hg-treated animals. Since neonatal CH3-Hg produces heart and kidney overgrowth, we examined the potential role of sympathetic input in altered tissue growth, utilizing chemical sympathectomy with 6-hydroxydopamine (6-OHDA). Sympathectomy inhibited the early phase of renal overgrowth, suggesting that sympathetic nerves participate in the initial effect of CH3-Hg on this tissue; however, 6-OHDA did not influence later phases of renal enlargement nor did it alter the CH3-Hg-induced cardiac overgrowth. These results indicate that neonatal exposure to CH3-Hg alters the synaptic development of peripheral catecholamine neurons, which may play a role in some of the subsequent effects on tissue development.     

Dermorphin and related peptides in rat tissues

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a naturally occurring peptide isolated from arboreal frog skin, is endowed with outstanding structural and biological features. It has no structural community with the sequence of mammalian opioid peptides and is a unique example of a D-aminoacid containing peptide which is synthesized via ribosomal route. Dermorphin is the most potent of the opioid peptides or opiates in producing long lasting analgesia and catalepsy. Since most amphibians' secretory peptides have counterparts in the mammalian central nervous system and gastrointestinal tract, we have developed a sensitive enzyme immunoassay that can detect 1 pg dermorphin to verify the possibility of dermorphin or dermorphin-related peptides occurrence in mammalian tissues. Dermorphin-related peptides were purified by fast protein liquid chromatography followed by reverse phase high pressure liquid chromatography. Identification was achieved by chromatographic comparison with synthetic standards and immunological analysis. A peptide behaving like authentic dermorphin was detected (2 ng/g) in rat small intestine. Immunoreactive species of higher Mr were also detected in the brain, adrenal glands and gastrointestinal tract, they may represent extended forms of dermorphin or homologous peptides.     

Locus coeruleus neurons and sympathetic nerves: Activation by cutaneous sensory afferents

The effects of mechanical and thermal cutaneous sensory stimulation on the activity of central norepinephrine (NE) neurons in the locus coeruleus (LC) and on peripheral sympathetic nerve activity (NE-SNA) in a renal branch of the splanchnic nerve were studied, using electrophysiological techniques in the anesthetized rat. Noxious, mechanical sensory stimulation caused a parallel and virtually identical change in central NE-LC activity and peripheral NE-SNA and both neuronal systems showed a biphasic excitation-inhibition response. Both non-noxious and noxious thermal sensory stimulation above 36 degrees C caused a parallel increase in NE-LC firing rate and NE-SNA, changes that were accompanied by increases in arterial blood pressure and heart rate. The increases in central and peripheral NE neuronal activity were linearly correlated. However, the two neuronal systems differed in their ability to adapt during prolonged noxious thermal stimulation. NE-LC neurons adapted completely and returned to baseline firing rate within 5 min during ongoing noxious thermal stimulation. In contrast, an increase in NE-SNA remained throughout the stimulation period. In recent studies in this laboratory, blood volume depletion was found associated with a marked NE-LC activation. In contrast to the present results during prolonged noxious stimulation, there was no attenuation of the NE-LC activation during prolonged hemorrhage. Taken in conjunction, these data indicate a discriminatory capacity of the NE-LC system in monitoring sensory or autonomic stimuli. Thus, only those stimuli of imperative importance for the animal were found to be associated with continuing robust NE-LC discharge.     

Mu receptors at discrete hypothalamic and brainstem sites mediate opioid peptide-induced increases in central sympathetic outflow

Synthetic human beta-endorphin, 7.25 nmol intracisternally, in conscious, freely moving, cannulated adult male rats increased plasma concentrations of the 3 catecholamines, epinephrine, norepinephrine and dopamine. Similarly administered equimolar morphine increased only plasma epinephrine concentration significantly. A 10-fold greater intracisternal dose of morphine significantly increased plasma concentrations of all 3 catecholamines. This effect was inhibited by prior intra-arterial naloxone administration. Intracisternal administration of the selective mu receptor agonist [D-Ala2,NMe-Phe4,Gly-ol5]enkephalin (DAGO), 2.9 nmol, also increased plasma concentrations of the 3 catecholamines and, furthermore, these effects were significantly greater than those noted in response to equimolar beta-endorphin. The greater potency of DAGO than beta-endorphin to increase catecholamine secretion suggests that this opioid peptide-induced effect is mediated at mu receptors. Administration of DAGO, 0.1 nmol, directly into either the hypothalamic paraventricular nucleus (PVN) or brainstem nucleus of the solitary tract (NTS) significantly increased plasma concentrations of all 3 catecholamines when compared with either saline-infused controls or animals administered DAGO into other brain areas. These catecholamine-stimulating effects of DAGO administered into either PVN or NTS were prevented by prior intra-arterial naloxone administration. Heart rate, but not mean arterial blood pressure, increased in response to DAGO administration into the NTS while no significant cardiovascular changes were noted among the experimental groups in response to DAGO administered into the PVN. These data support a hypothesis that mu receptors at discrete and anatomically distant brain sites mediate opioid peptide-induced catecholamine secretion through activation of the central sympathetic outflow to the adrenal medulla and sympathetic nerve terminals.     

Effects of adrenalectomy, propanolol and methylatropine on the increase in heart rate induced by injection of dermophin in the rat anterior hypothalamic nucleus

Anterior hypothalamic injections of 40 pmol dermophin, a potent opiate receptor agonist, increased heart rate 17% and had no effect on blood pressure in halothane-anesthetized rats. Administration of the beta-receptor antagonist, propanolol, during the peak response to dermorphin, reduced the heart rate to levels not different from pretreatment control; pretreatment with propranolol completely blocked the tachycardia produced by a subsequent injection of dermorphin. In contrast, neither adrenalectomy nor pretreatment with methylatropine altered the response to dermorphin. These data suggest that increased activity of the sympathetic nervous system, primarily to the heart, and not increased release of adrenal catecholamines or inhibition of parasympathetic nervous system activity, is responsible for the increase in heart rate resulting from injection of dermorphin into the hypothalamic nuclues.     

Urinary excretion of catecholamines and their metabolites in relation to circulating catecholamines. Six-hour infusion of epinephrine and norepinephrine in healthy volunteers.

Some depressed patients have been shown to excrete abnormal amounts of catecholamines and their metabolites in urine. Some studies suggest that hypersecretion of epinephrine by the adrenals and of norepinephrine by the peripheral sympathetic system cause increased excretion of urinary catecholamines and their metabolites in a subgroup of patients. To evaluate the effect of increased catecholamine levels in the peripheral circulation on urinary catecholamine and metabolite levels, we infused healthy volunteers during 6 hours with epinephrine, norepinephrine, or placebo, respectively, in a three-period, double-blind, crossover design. The results indicate that (1) urinary epinephrine and norepinephrine levels were the most sensitive indicators of increased circulating epinephrine and norepinephrine levels, respectively; (2) changes in circulating epinephrine or norepinephrine levels were not readily reflected in changes in urinary vanillylmandelic acid or 3-methoxy-4-hydroxyphenylglycol levels; and (3) increased normetanephrine excretion was not only induced by infusion of norepinephrine but also by epinephrine. This last finding may be due to activation of the sympathetic nervous system by circulating epinephrine. These results may help to explain the mechanism of adrenal epinephrine and sympathetic nervous system norepinephrine hypersecretion observed in subgroups of depressed patients.     

Basal sympathoadrenal function in posttraumatic distress disorder.

Research has consistently shown that patients with posttraumatic stress disorder (PTSD) manifest greater changes in heart rate, blood pressure, and plasma epinephrine than controls when exposed to trauma-related laboratory stressors. However, findings are equivocal as to whether PTSD subjects differ from controls on basal, or tonic, measures of autonomic activity. In this study, PTSD patients (n = 11) and asymptomatic controls (n = 11) were compared on measures of basal sympathoadrenal function, including plasma norepinephrine and epinephrine as well as heart rate and blood pressure. Results showed that PTSD patients were not significantly different from control subjects on any measure. Although phasic alterations in autonomic function in PTSD have been consistently found in previous research, this study suggests that tonic sympathetic nervous system activity in PTSD patients may not differ from that of healthy controls.     

Peripheral sympathectomy and adrenal medullectomy do not alter cerebrospinal fluid norepinephrine

Despite a blood-brain barrier for norepinephrine, the concentration of norepinephrine in plasma and cerebrospinal fluid has been observed to be similar. This relationship between plasma and cerebrospinal fluid norepinephrine levels suggest that peripheral sympathetic neurons innervating blood vessels to brain and spinal cord may contribute significantly to cerebrospinal fluid norepinephrine levels, and questions the validity of cerebrospinal fluid norepinephrine as an index of central nervous system noradrenergic activity. We demonstrate that extensive destruction of the peripheral sympathetic nervous system and the adrenal medulla has no effect on rat cerebrospinal fluid norepinephrine. It is therefore unlikely that peripheral sources of norepinephrine contribute significantly to cerebrospinal fluid norepinephrine levels.     

Effects of lithium administration on plasma catecholamines

The mode of action of lithium in the treatment of bipolar affective disorder is unknown. Among its actions are effects on release and response to catecholamines in experimental animals. We have therefore examined its effect on peripheral catecholamines in man. After 3 weeks of administration of therapeutic doses of lithium carbonate, healthy volunteers showed a differential response of catecholamines to insulin stimulation. Although the response of plasma norepinephrine remained unchanged, epinephrine response was dramatically reduced. These findings provide additional evidence for the separate neural regulation of the adrenal medulla and sympathetic nerve endings. Further studies are necessary to determine whether these effects on peripheral epinephrine are paralleled by changes in central epinephrine.     

Reflex changes in sympatho-adrenal medullary functions in response to baroreceptor stimulation in anesthetized rats

Effects of increased and decreased blood pressure on adrenal sympathetic nerve activity and catecholamine secretion were examined in anesthetized Wistar rats. Efferent nerve impulses were counted and adrenal venous plasma was collected just before and 30 s after the onset of hypotensive stimulation induced by bleeding (average decrease in systolic blood pressure over the stimulation period was 38 mm Hg) or a hypertensive stimulation induced by phenylephrine administration (average increase in systolic blood pressure over the stimulation period was 41 mm Hg). While nerve activity and epinephrine and norepinephrine secretion were not significantly altered by the change in blood pressure in baroreceptor denervated animals, there were increases in these variables (131.1% of control levels for epinephrine, 117.5% for norepinephrine, and 127.1% for nerve activity) in response to bleeding and a marked decrease (to 63.0% of control levels for epinephrine, 53.9% for norepinephrine, and 54.0% for nerve activity) in response to phenylephrine administration with baroreceptors intact. These changes were significant both with respect to control blood pressure levels and with regard to the effect of stimulation in the baroreceptor-denervated animals. The reflex responses in adrenal nerve and catecholamine secretion were generally proportional in magnitude. The present results are discussed with regard to the effects of somatic stimulation on nerve activity and catecholamine secretion.     

Cardiovascular effects of centrally administered vasopressin in conscious and anesthetized rats

Intracerebroventricular (ICV) injections of arginine vasopressin (AVP) in doses of 0.015 nmoles and 0.15 nmoles produced a fall in mean actual pressure heart rate and respiration in pentobarbital anesthetized rats. The changes in mean arterial pressure and respiration after the higher dose were significantly different from saline injection. In contrast, the same doses of AVP given to conscious animals increased both blood pressure and heart rate. Following the 0.15 nmole dose, there was a marked and significant rise in plasma norepinephrine and epinephrine, indicating that activation of the sympathetic nervous system was, at least in part, responsible for the rise in blood pressure. Plasma vasopressin increased by less than 10 pg/ml following injection. Similar doses of a vasopressin pressor antagonist had no significant effect on mean arterial pressure or heart rate. These results indicate that ICV injection of vasopressin has different effects on blood pressure, depending on the presence or absence of anesthesia: depressor responses in the anesthetized animal and pressor responses in the unanesthetized animal.     

Proenkephalin-A derived peptides do not modulate cardiovascular effects of epinephrine on the isolated rat atrial preparations

The catecholamines and the opioid peptides are found to be co-localized in the adrenomedullary chromaffin cells. They are co-secreted from the chromaffin granules in response to various stimuli. The stress-induced released of epinephrine is known to exert its effect on the cardiovascular system resulting in the changes in heart rate and blood pressure. However, the role of the co-released proenkephalin-A derived peptides has not been extensively characterized. Previous work from several investigators suggested that the peptides modulate cardiac functions of the catecholamines. There is considerable conflicting results among these reports. Results from the isolated rat atrial preparation indicated that enkephalins attenuated the increase in atrial rate induced by norepinephrine through restriction of the calcium fluxes. Nonetheless, others reported insensitivity of the enkephalins in similar or different test systems. We further re-examined these discrepancies using the isolated rat atrial preparation to investigate the opioid peptide modulatory effect on the cardiovascular changes induced by exogenous epinephrine. Alterations in rate and force of contraction resulting from epinephrine and the peptides were both studied in parallel. The opioid peptides used in this study were [Met5]-enkephalin (ME), [Leu5]-enkephalin (LE), FMRFamide, [Met5]-enkephalyl-Arg6-Phe7 (MEAP), peptide E, and the non-selective opioid agonist, etorphine. We report here that none of the opioid peptides were effective in alleviating or attenuating the increase in heart rate and developed tension caused by epinephrine. The peptides did not affect the basal beating rate nor the force of contraction. Thus, the present results clearly demonstrate the insensitivity of the enkephalins in modulating the cardiac effects of epinephrine. They further indirectly support the prejunctional synaptic nerve endings as the potential peripheral site of action of the peptides.