Β-adrenoceptor Responsiveness Research Articles

β2 -Adrenoceptors indirectly support impaired β1 -adrenoceptor responsiveness in the isolated type 2 diabetic rat heart.

What is the central question of this study? Are there specific contributions of β1 - and β2 -adrenoceptor subtypes to the impaired β-adrenoceptor responsiveness of the type 2 diabetic heart? What is the main finding and its importance? In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the β1 -adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the β2 -adrenoceptor subtype actions seem to support function in the diabetic heart indirectly. Impaired β-adrenoceptor (β-AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of β1 - and β2 -AR subtypes to β-AR-associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific β1 - and β2 -AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff-perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the β-AR agonist isoprenaline (1×10-11 to 3×10-8 moll-1 ) with or without the selective β1 -AR antagonist CGP20712A (3×10-8 moll-1 ) or the β2 -AR antagonist ICI-118,551 (5×10-8 moll-1 ), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non-diabetic 216±17beatsmin-1 versus diabetic 151±23beatsmin-1 , P<0.05). However, the β-AR-induced increase in HR was similar and was completely blocked by the β1 -AR antagonist, but not by the β2 -AR antagonist. The β-AR-induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the β1 -AR antagonist and partly impaired by the β2 -AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non-diabetic 1.62±0.19a.u. versus diabetic 2.30±0.25a.u., P<0.05). In conclusion, the β1 -AR is the main subtype regulating chronotropic, inotropic and lusitropic β-AR responses in the healthy heart and the type2 diabetic heart. The β2 -AR subtype indirectly supports the β1 -AR functional response in the diabetic heart. This suggests that β2 -ARs could be an indirect target to improve the function of the heart in type 2 diabetes.

β1 -Adrenoceptor, but not β2 -adrenoceptor, subtype regulates heart rate in type 2 diabetic rats in vivo.

What is the central question of the study? The sympathetic system regulates heart rate via β-adrenoceptors; this is impaired during diabetes. However, the specific β-adrenoceptor subtype contributions in heart rate regulation in diabetes in vivo are unknown. What is the main finding and its importance? Telemetric recordings in conscious non-diabetic and type 2 diabetic rats demonstrated that the β1 -adrenoceptor subtype, and not the β2 -adrenoceptor, regulated the lower resting heart rate and increased β-adrenoceptor responsiveness in diabetes in vivo. This provides new physiological insight into the dysregulation of heart rate in type 2 diabetes, which is important for improving therapeutic strategies targeting the diabetic chronotropic incompetence. β-Adrenoceptor blockers are widely used to reduce heart rate, the strongest predictor of mortality in cardiac patients, but are less effective in diabetic patients. This study aimed to determine the specific contributions of β1 - and β2 -adrenoceptor subtypes to chronotropic responses in type 2 diabetes in vivo, which are currently unknown. Type 2 diabetic and non-diabetic rats were implanted with radiotelemeters to measure arterial blood pressure and derive heart rate in conscious conditions. Vascular access ports were implanted to inject isoprenaline (β1 - and β2 -adrenoceptor agonist, 0.1-300μgkg-1 ) in the presence of atenolol (β1 -adrenoceptor antagonist, 2000μgkg-1 ) or nadolol (β1 - and β2 -adrenoceptor agonist, 4000μgkg-1 ) to determine the chronotropic contributions of the β-adrenoceptor subtypes. Resting heart rate was reduced in diabetic rats (388±62 versus 290±37beatsmin-1 non-diabetic versus diabetic, P<0.05, mean±SD), which remained after atenolol or nadolol administration. Overall β-adrenoceptor chronotropic responsiveness was increased in diabetic rats (change in heart rate at highest dose of isoprenaline: 135±66 versus 205±28beatsmin-1 , non-diabetic versus diabetic, P<0.05), a difference that diminished after β1 -adrenoceptor blockade with atenolol (change in heart rate at highest dose of isoprenaline: 205±37 versus 195±22beatsmin-1 , non-diabetic versus diabetic, P<0.05). In conclusion, the β1 -adrenoceptor is the main subtype to modulate chronotropic β-adrenoceptor responses in healthy and diabetic rats. This study provides new insights into the pathological basis of dysregulation of heart rate in type 2 diabetes, which could be important for improving the current therapeutic strategies targeting diabetic chronotropic incompetence.

Chronic bilateral renal denervation reduces cardiac hypertrophic remodelling but not β-adrenergic responsiveness in hypertensive type 1 diabetic rats.

What is the central question of this study? Can bilateral renal denervation, an effective antihypertensive treatment in clinical and experimental studies, improve cardiac β-adrenoceptor responsiveness in a diabetic model with underlying hypertension? What is the main finding and its importance? Bilateral renal denervation did not affect β-adrenergic responsiveness in the diabetic hypertensive rat heart, but denervation reduced the hypertension-induced concentric hypertrophic remodelling. This suggests that the positive haemodynamic changes induced by renal denervation are most likely to reflect an attenuation of sympathetic effects on the systemic vasculature and/or the renal function rather than direct sympathetic modulation of the heart. Bilateral renal denervation (BRD) has been shown to normalise blood pressure in clinical and experimental studies of hypertension by reducing systemic sympathetic output. This study determined the effect of BRD on cardiac β-adrenoceptor (AR) responsiveness in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. Bilateral renal denervation or sham surgeries were conducted repeatedly at 3, 6 and 9weeks in Ren-2 rats with or without streptozotocin (STZ)-induced diabetes (4×n=7); Sprague-Dawley rats (n=6) served as control animals. Cardiac function was determined in isolated hearts at 18weeks of age. Normalised left ventricular developed pressure and relaxation was recorded in response to incremental concentrations of the β-AR agonist isoprenaline (from 10-10 to 10-7m) or the β3 -AR agonist BRL37344 (from 10(-13) to 10(-6) m). Expression levels of β1 -AR were determined by Western blot. Both inotropic and lusitropic β-AR responsiveness was reduced in the hypertensive diabetic hearts, but these responses were unaltered after BRD. Expression levels of β1 -AR were increased after BRD (Sham, 0.85±0.11versus 1.01±0.05a.u.; BRD, 1.45±0.11versus 1.46±0.07a.u.; Ren-2versus Ren-2 STZ, P<0.05versus Sham). No effect of β3 -AR agonist stimulation with BRL37344 was observed. Interestingly, BRD increased left ventricular diastolic volume in both the Ren-2 and the Ren-2 STZ groups. Bilateral renal denervation did not restore the attenuated cardiac β-AR responsiveness in the diabetic hypertensive rats, but it reduced the extent of hypertension-induced concentric hypertrophic remodelling. Thus, the haemodynamic protection offered by renal denervation appears to reflect an attenuated sympathetic innervation of the systemic vasculature and/or kidney rather than a direct cardiac effect.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses

To study the consequences of glucocorticoid treatment in fetal growth restriction (FGR) on cardiac function. Laboratory. Sheep. Growth restriction was induced in sheep fetuses using single umbilical artery ligation (SUAL) on days 105-110 of gestation (term 147). Control fetuses were not ligated. Betamethasone (BM) (11.4 mg intramuscularly) or saline was administered to ewes on days 5 and 6 after surgery. Ewes were anaesthetised on day 7, the fetuses were removed, and their hearts were mounted on a Langendorff apparatus. Balloon catheters were inserted into the right and left ventricles. Ventricular contractile function and infarct area following ischaemia/reperfusion. The SUAL resulted in FGR (body weight 77% of control). The FGR was associated with increases in basal left ventricular pressure development and rates of contraction and relaxation. Right ventricular contraction was unaffected. Following brief ischaemia/reperfusion, the infarct area in FGR hearts was increased four-fold compared with controls. Antenatal BM resulted in a proportional increase in heart size and coronary flow, especially in FGR fetuses, and left ventricular pressure and heart rate responses to β-adrenoceptor activation were increased. Fetal hearts rapidly adapt to FGR to maintain substrate delivery to the brain and heart. The FGR greatly enhanced the area of ischaemia, with implications for susceptibility in postnatal life. Antenatal BM treatment does not interfere with these cardiac changes but appears to increase left ventricle β-adrenoceptor responsiveness, which may render the offspring vulnerable to subsequent cardiac dysfunction.

Regulation of Sympathetic and Parasympathetic Receptor Responses in the Rat Trachea by Epithelium: Influence of Mechanical and Chemical Removal of Epithelium

Removal of the epithelial layer of rat tracheal tissue did not affect the methacholine-induced contraction of the tracheal smooth muscle, but attenuated the (-)-isoprenaline induced relaxation (expressed as percentage of the methacholine contraction). In this way the epithelial layer seemed to play a role in the maintenance of an autonomic balance between sympathetic and parasympathetic receptor responses. Incubation of rat tracheal tissue with cumene hydroperoxide (3 x 10(-5)-10(-3) M) resulted in a dose-dependent destruction and (partial) removal of the epithelial layer. Cumene hydroperoxide diminished muscarinic receptor responses of the rat trachea. Moreover, the autonomic balance between muscarinic and beta-adrenoceptor responses was affected. The effects of cumene hydroperoxide on receptor responses were more pronounced after epithelium removal. The protective role of the epithelial layer of pulmonary tissue against oxidative stress has therefore been emphasized.

Affinity constants and beta-adrenoceptor reserves for isoprenaline on cardiac tissue from normotensive and hypertensive rats.

To determine whether there are differences in cardiac beta-adrenoceptor responsiveness, isoprenaline affinity constants and fractional beta-adrenoceptor occupancy-response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-beta-(2-hydroxyl-3-alpha-naphthoxypropy lamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible beta-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD2 values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline KA values were 2-3 x 10(-6) M, which is as expected for isoprenaline at beta1 or beta2-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline KA values were 2-4 x 10(-8) M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3-4% of the beta-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25-35% and 55%, respectively, of the beta-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller beta-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline KA values on the WKY right atrium. The isoprenaline KA value on the SHR right atrium was 1-4 x 10(-8) M. Because the isoprenaline KA values for the left and right atria are markedly different from those previously reported for isoprenaline at beta1 or beta2-adrenoceptors, we suggest that atypical beta-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower beta-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.

The influence of cellular metabolism on the - and -adrenoceptor responses of the rabbit small intestine.

The influence of cellular metabolism on the - and -adrenoceptor responses of the rabbit small intestine.

Modulation of intracellular calcium transient in response to β-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Importance of adrenergic receptors in prenatally induced cognitive impairment in the domestic chick

In the domestic chick, mild hypoxia (24h of 14% oxygen) at two stages of embryonic development results in post-hatch memory deficiencies tested using a discriminated bead avoidance task. The nature of the memory loss depends on the gestational age at which the hypoxia occurs. Hypoxia on embryonic day 10 (E10) of a 21 day incubation results in chicks with no short-term memory 10min after training, whereas hypoxia on day 14 (E14) results in chicks with good labile memory 30min after training but no consolidation of memory into permanent storage (120min). Hypoxia at E14 is associated with increased plasma levels of noradrenaline and therefore we suggest that altered catecholamine exposure within the brain contributes to cognitive problems by modifying the responsiveness of brain β-adrenoceptors. In ovo administration of noradrenaline, or the β2-adrenoceptor agonist formoterol, at E14 had the same effect on memory consolidation as hypoxia. Following hypoxia at E14, memory could be rescued after training by central injection of a β3-adrenoceptor agonist, but not by a β2-adrenoceptor agonist. The differences in the responsiveness of memory processing to β2-adrenoceptor agonists suggests alterations to the receptors or downstream of the receptor activation. However, both types of β-adrenoceptor agonists rescued memory in E10 treated chicks implying that at this age hypoxia does not affect the receptors. In summary, hypoxia or increased levels of stress hormones during incubation alters β-adrenoceptor responsiveness; the outcome of the insult depends upon the cellular developmental processes at a given embryonic stage.

Changes in cardiovascular β-adrenoceptor responses during hypothermia

The purpose of this study was to determine cardiovascular β-adrenergic responses during hypothermia. In the present study, we used isoproterenol (Iso), a nonselective, potent β-adrenoceptor agonist, well known for its positive chronotropic and inotropic pharmacologic actions at normothermia. Rats were instrumented to measure mean arterial pressure (MAP) and left ventricular (LV) pressure–volume changes using a Millar pressure–volume conductance catheter. Core temperature was manipulated from 37 (normothermia) to 24 °C (hypothermia) and back to 37 °C (rewarming) using both internal and external heat exchangers. During cooling at each temperature (33, 30, 27, and 24 °C), central hemodynamic variables and MAP were measured while intravenously infusing Iso (doses of 1.7, 5, 10, and 20 ng/min). Seven animals underwent all phases of the protocol. At normothermia Iso infusion resulted in a significant, dose-dependent increase in heart rate (HR), stroke volume (SV), cardiac output (CO), LV d P/d t max (left ventricular maximum derivative of systolic pressure over time) but no change in MAP. During cooling Iso infusion caused no dose-dependent change in any of the hemodynamic variables. After rewarming, baseline HR and LV d P/d t max were increased, whereas SV was significantly reduced when compared with their pre-hypothermic baseline values. This study shows that physiological cardiovascular responses mediated by the β-adrenoceptor are significantly diminished during core hypothermia.

The role of Gi proteins in reduced vasorelaxation response to β-adrenoceptor agonists in rat aorta during maturation

β-adrenoceptor mediated vasorelaxation and cAMP production decline during maturation and aging in rat aorta. β-adrenoceptor-stimulated vasorelaxation is mainly triggered by Gsα-mediated activation of adenylyl cyclase. β2-adrenoceptors can also activate Gi protein which inhibits adenylyl cyclase activity. In this study, we examined the role of Gi proteins in the decreased β-adrenoceptor mediated responses during maturation. Pertussis toxin treatment of aortic rings to inhibit Giα activation completely restored age related decline in isoproterenol-stimulated maximal vasorelaxation in 3-month old rats. This treatment increased the potency, but not the maximal response of isoproteronol to produce vasorelaxation in 6 month old rats. The maximal isoproteronol stimulated cAMP responses were also partially restored in pertussis toxin-treated rings from 3 or 6-month old rats. We also examined β-adrenoceptor stimulated binding of 35[S]GTPγS to Gsα and Giα1/2 in aortic membranes from 1, 3 and 6-month old rats. In 1-month old rats, isoproterenol-stimulated 35[S]GTPγS binding to Gsα was significantly higher than that of 3 or 6-month old rats. Isoproterenol-stimulated 35[S]GTPγS binding to Giα1/2 was found to be significantly increased in 3 or 6-month old rats compared to 1-month old rats. The results of this study showed that β-adrenoceptor-mediated activation of Gs and Gi proteins was declined and increased, respectively, and inhibition of the Gi mediated activity by pertussis toxin treatment partially restored impaired vasorelaxation and cAMP response to β-adrenoceptor stimulation during maturation in rat aorta. The decrease in β-adrenoceptor mediated activation of Gs gradually increased during maturation. All together these results indicated that β-adrenoceptor mainly activates Gs protein in aorta from 1-month old rats, while it activates Gi and with a certain degree of decline it also activates Gs in aorta from 3 and 6-months old rats and not only the increase in β-adrenoceptor coupling to Gi but also the decrease in its coupling to Gs play a role in the impaired β-adrenoceptor responses in rat aorta during maturation.

Alterations of β-adrenoceptor responsiveness in postischemic myocardium after 72 h of reperfusion

To determine the effect of a completely developed reperfused myocardial infarction model on β-adrenoceptor responsiveness, we induced a 90-min regional ischemia followed by 72 h of reperfusion in dog hearts. Regional myocardial blood flow was determined after 60 min of ischemia using radioactive microspheres. β-adrenoceptor density was reduced in the ischemic endocardium (95±16 fmol/mg) and epicardium (160±13 fmol/mg) compared to the nonischemic region (304±21 fmol/mg). β-adrenoceptor density in the ischemic endocardium varied with the degree of collateral blood flow measured ( r 2=0.79, P<0.05); this relation was the opposite of that in the ischemic epicardium ( r 2=0.77, P<0.05). Higher levels of tissue catecholamines and G protein-coupled receptor kinase 2 (GRK2) were observed in the ischemic epicardium as compared to nonischemic tissue. Forskolin-induced adenylyl cyclase activities were depressed in both ischemic regions as compared to nonischemic region, correlating with a reduction in regional myocardial blood flow. Using forskolin stimulation as covariate, no difference in isoproterenol-induced adenylyl cyclase activity was identified in the different regions. It is concluded that cAMP production induced by β-adrenoceptor activation is dependent upon adenylyl cyclase enzyme activity rather than β-adrenoceptor density in the ischemic myocardium. However, the density of the β-adrenoceptor in the viable ischemic regions can be modified by the presence of GRK2 and tissue catecholamines, an index of regional sympathetic efferent postganglionic nerve terminal activity.

Loss of β-adrenoceptor response in myocytes overexpressing the Na +/Ca 2+-exchanger

Increased Na +/Ca 2+-exchanger (NCX) and altered β-adrenoceptor (βAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the β 1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration–response curve. Responses to isoproterenol could be restored to normal by β 2AR blockade, suggesting a β 2AR-mediated inhibition of β 1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that β 2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to β 2AR–Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of βAR stimulation and that this may contribute to the alterations in βAR response seen in failing human heart.

Characterization of cardiac hypertrophy and heart failure due to volume overload in the rat.

Alterations in general characteristics and morphology of the heart, as well as changes in hemodynamics, myosin heavy chain isoforms, and beta-adrenoceptor responsiveness, were determined in Sprague-Dawley rats at 1, 2, 4, 8, and 16 wk after aortocaval fistula (shunt) was induced by the needle technique. Three stages of cardiac hypertrophy due to volume overload were recognized during the 16-wk period. Developing hypertrophy occurred within the first 2 wk after aortocaval shunt was induced and was characterized by a rapid increase of cardiac mass in both left and right ventricles. Compensated hypertrophy occurred between 2 and 8 wk after aortocaval shunt where normal or mild depression in hemodynamic function was observed. Decompensated hypertrophy or heart failure occurred between 8 and 16 wk after aortocaval shunt and was characterized by circulatory congestion, decreased in vivo and in vitro cardiac function, and a shift in myosin heavy chain isozyme expression. However, the positive inotropic effect of isoproterenol was augmented at all times during the 16-wk period. Characterization of beta-adrenoceptor binding in failing hearts at 16 wk revealed a significant increase in beta(1)-receptor density, whereas beta(2)-receptor density was unchanged. Consistent with this, basal adenylyl cyclase activity was significantly increased, and both isoproterenol- and forskolin-stimulated adenylyl cyclase activities were also increased. These results indicate that upregulation of beta-adrenoceptor signal transduction is a unique feature of cardiac hypertrophy and failure induced by volume overload.

Cardiac β-Adrenoceptor Desensitization Due to Increased β-Adrenoceptor Kinase Activity in Chronic Uremia

ABSTRACT. Patients with chronic renal failure develop an autonomic dysfunction with impaired baroreflex control and attenuated cardiovascular β-adrenoceptor response to noradrenaline. In rats that underwent 5/6-nephrectomy (SNX), cardiac β-adrenoceptor responsiveness was reduced as well. Therefore, the aim of this study was to further investigate the mechanism underlying cardiac β-adrenoceptor desensitization in SNX rats. For this purpose, right and left ventricular β-adrenoceptor density, activity of the G-protein–coupled receptor kinase, and activity and density of the neuronal noradrenaline transporter (uptake1) were assessed in SNX rats. Seven weeks after SNX, rats had developed left heart hypertrophy. Plasma creatinine, urea, and noradrenaline levels were significantly increased; left and right ventricular noradrenaline content was significantly decreased when compared with sham-operated control rats. In these SNX rats, left, but not right, ventricular β-adrenoceptor density was significantly reduced, and membrane-associated G-protein–coupled receptor kinase activity was significantly increased compared with sham-operated rats. Although right and left ventricular activity of uptake1was unchanged, the neuronal noradrenaline transporter density was significantly reduced in both ventricles of SNXversussham-operated rats. An increase in left ventricular G-protein–coupled receptor kinase activity, possibly triggered by enhanced cardiac noradrenaline release, might be responsible for the decrease in left ventricular β-adrenoceptor responsiveness in SNX rats.

Estrogen Modulates α1/β-Adrenoceptor- Induced Signaling and Melatonin Production in Female Rat Pinealocytes

Nocturnal rise in pineal melatonin output is due to the night-induced acceleration of noradrenergic transmission and α₁- and β-adrenoceptor activation. In addition, in female animals, cyclic oscillations in circulating levels of sex steroid hormones are accompanied by changes in the rate of pineal melatonin secretion. To investigate whether estrogen directly affects pineal adrenoceptor responsiveness, pinealocytes from 21-day-old ovariectomized rats were exposed to physiological concentrations of 17β-estradiol (17β-E₂) and treated with noradrenergic agonists. Direct exposure to 17β-E₂ reduced α₁/β-adrenoceptor-induced stimulation of melatonin synthesis and release. This effect was mediated by an estrogen-dependent inhibition of both β-adrenoceptor-induced accumulation of cAMP and α₁-adrenoceptor-induced phosphoinositide hydrolysis. Furthermore, estrogen reduced transient Ca²⁺ signals elicited in single pinealocytes by α₁-adrenoceptor activation or by potassium-induced depolarization. In the case of β-adrenoceptor responsiveness, neither forskolin- nor cholera toxin-induced accumulation of cAMP were affected by previous exposure to 17β-E₂. This indicates that estrogen effects must be exerted upstream from adenylylcyclase activation, and independent of modifications in G protein expression, therefore suggesting changes in either adrenoceptor expression or receptor-effector coupling mechanisms. Since estrogen effects upon adrenoceptor responsiveness in pineal cells was not mimicked by 17β-E₂ coupled to bovine serum albumin and showed a latency of 48 h, this effect could be compatible with a genomic action mechanism. This is also consistent with the presence of two estrogen receptor proteins, α- and β-subtypes, in female rat pinealocytes under the present experimental conditions.

Ontogeny of G-protein expression: control by β-adrenoceptors

Cardiac cell homeostasis is maintained in the face of excessive β-adrenoceptor stimulation through the process of desensitization. Desensitization is not an inherent property of these cells but rather is acquired during development; neonates given β-agonists actually show heterologous sensitization, involving changes in the expression and catalytic activity of adenylyl cyclase (AC) as well as an increased receptor/G-protein coupling. The current study examines the role of specific G-protein components, G sα and G iα, in the ontogeny of β-adrenoceptor responses and in the transition from agonist-induced sensitization to desensitization. Between postnatal days (PN) 6 and 15 there was a significant decrease in the 52 kDa isoform of G sα with no accompanying change of the 45 kDa form; over the same period, G iα3 also declined substantially. In contrast, the 45 kDa isoform of G sα and G iα1,2 remained fairly constant over the same period and fluoride-stimulated AC activity increased. Treatment with isoproterenol on PN2–5 did not result in any significant changes in G sα expression but robustly decreased G iα1,2. These changes were accompanied by heterologous sensitization of AC activity at the level of AC itself, evidenced by equivalent increases in the enzymatic response to fluoride and forskolin–Mn 2+. Isoproterenol given to older animals (PN11–14) also caused specific loss of G i protein, in this case targeting G iα3, whereas G sα again was unchanged; in contrast to the younger group, the older animals displayed heterologous desensitization of AC at the level of G-protein function (specific loss of the fluoride response). These results indicate that the normal ontogenetic increase of cardiac β-adrenoceptor coupling to AC is not dependent on the absolute amount of G-proteins, nor on the relative balance of stimulatory (G s) and inhibitory (G i) subunits. However, the ability of receptor stimulation to downregulate G iα1,2, an event which is specific to immature cardiac cells, is likely to be an important component of the resistance of the fetal/neonatal heart to agonist-induced desensitization and hypertrophy. The maintenance of cardiac β-adrenoceptor signaling in the face of intense stimulation is likely to play an important role in the physiologic adaptations necessary to the perinatal transition.

Depression of the inotropic action of isoprenaline by nitric oxide synthase induction in rat isolated hearts

The mechanisms involved in myocardial dysfunction during septic shock are not well understood. We have investigated the effects of endotoxin and the role of nitric oxide (NO) in the β-adrenoceptor responsiveness of rat isolated, ejecting hearts perfused at 60 mmHg of head pressure. In vivo pretreatment with endotoxin (4 mg/kg, i.p., 3 h before heart isolation) significantly attenuated the inotropic response (increase in left ventricular developed pressure, LVP) to isoprenaline (0.15 μg) after 30 min equilibration and after a further 90 min of perfusion. The peak rate of LVP development (d P/d t max) in response to isoprenaline was reduced by endotoxin pretreatment, as was the increase of coronary flow. The depression of ventricular contraction was prevented by pretreatment with dexamethasone (1 mg/kg, i.p., 30 min before endotoxin), and was also restored by perfusion with N G-nitro- l-arginine ( l-NA, 10 μM) for 60 min, but not by N G-nitro- d-arginine ( d-NA, 10 μM). Mercaptoethylguanidine (MEG, 30 μM), a selective inhibitor of the inducible NO synthase (isoform 2), also reversed the depression of the isoprenaline response caused by endotoxin pretreatment. However, treatment with endotoxin, dexamethasone, l-NA, d-NA or MEG had minimal effects on the baseline parameters of LVP, d P/d t max and coronary flow, which all tended to decline over the 2 h perfusion period. Western blot analysis using an antibody to NO synthase (isoform 2, but not to isoform 3) revealed the induction of a protein corresponding to NO synthase 2 in the endotoxin-treated hearts but not in control hearts or those treated with dexamethasone or MEG. In summary, these results indicate that endotoxin depresses myocardial contractile function and reduces inotropic responsiveness to β-adrenoceptor activation. The effect of endotoxin on the inotropic response is mediated, at least in part, by products of an endogenous NO synthase that is suppressed by dexamethasone and a specific inhibitor of NO synthase (isoform 2).

Abnormalities of the myocytes in ischaemic cardiomyopathy.

Acute myocardial ischemia and subsequent reperfusion result in biochemical and ionic changes in cardiac myocytes which cause contracture of the muscle and a reduced contractile force. Whether changes observed in single myocytes isolated from ischaemic ventricles are a direct consequence of the acute insult, or develop more slowly due to subsequent alterations in load and neurohumoural environment, is controversial. Myocytes from ischemic hearts have a similar contraction amplitude to those from non-failing hearts at physiological or maximally activating levels of ca2+. This could be partly due to the method of cell selection, or could represent the detection of a population of myocytes that have recovered from the original insult. However, there are significant decreases in the velocities of contraction and, particularly, relaxation in myocytes from the ischaemic heart. These resemble alterations caused by anoxia/reperfusion, but similar changes have also been observed in non-ischaemic causes of heart failure. Responses of beta-adrenoceptor stimulation are reduced in single cells from the failing heart, and a post-receptor defect has also been detected. Treatment with pertussis toxin, which reduces the activity of the inhibitory guanine-nucleotide binding protein (Gi) was able to restore beta-adrenoceptor responses to normal. The hypothesis that alterations in the beta-adrenoceptor/Gi/cAMP pathway represent the response of the myocyte to continued exposure to noradrenaline, because of the high sympathetic drive in these patients, is supported by the strong parallels observed with catecholamine-treated animals, and by the fact that non-ischemic aetiologies exhibit similar desensitization. It is concluded that the surviving myocytes in an ischaemic heart are damaged by the neurohumoral alterations that represent the body's attempt to restore cardiac output.

Mechanism of protein kinase c potentiation of airway β-adrenergic relaxation

To evaluate the regulatory action of protein kinase C (PKC) on airway β-adrenergic function, the relaxant effects of isoproterenol (ISO) and 8 bromo-cyclic AMP (BrcAMP) were examined in tracheal smooth muscle (TSM) segments half-maximally contracted with acetylcholine in the absence (control) and presence of PKC activation with the phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPS). Relative to control tissues, TSM treated with 0.1μM DPB depicted significantly enhanced maximal relaxation and sensitivity to ISO but not to BrcAMP. The enhancing effect of DPB on ISO responsiveness was completely inhibited in the presence of the PKC antagonist H-7. Inhibition of the Na+-K+ pump with either ouabain or K+-free buffer diminished the TSM relaxant response to ISO but not to BrcAMP. Inhibition of the Na+-K+ pump also ablated the DPB-induced potentiation of β-adrenoceptor responsiveness. Collectively, these data demonstrate that: 1) PKC activation enhances TSM relaxant responsiveness to β-adrenoceptor stimulation; 2) inhibition of the airway Na+-K+ pump markedly blunts the relaxant response to β-adrenoceptor stimulation; and 3) inhibition of the Na+-K+ pump abolishes the above potentiating effect of DPB on β-adrenoceptor-mediated relaxation of rabbit TSM. Thus, the above findings provide new evidence that PKC activation enhances the airway relaxant response to β-adrenoceptor stimulation, and that the latter effect is dependent on potentiated stimulation of the airway electrogenic Na+-K+ pump.