nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure

We hypothesized that gene transfer of neuronal nitric oxide synthase (nNOS) into the rostral ventrolateral medulla (RVLM) improves baroreflex function in rats with chronic heart failure (CHF). Six to eight weeks after coronary artery ligation, rats showed hemodynamic signs of CHF. A recombinant adenovirus, either Ad.nNOS or Ad.beta-Gal, was transfected into the RVLM. nNOS expression in the RVLM was confirmed by Western blot analysis, NADPH-diaphorase, and immunohistochemical staining. We studied baroreflex control of the heart rate (HR) and renal sympathetic nerve activity (RSNA) in the anesthetized state 3 days after gene transfer by intravenous injections of phenylephrine and nitroprusside. Baroreflex sensitivity was depressed for HR and RSNA regulation in CHF rats (2.0 +/- 0.3 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 3.8 +/- 0.3 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01, respectively). Ad.nNOS transfer into RVLM significantly increased the HR and RSNA ranges (152 +/- 19 vs. 94 +/- 12 beats/min, P < 0.05 and 130 +/- 16 vs. 106 +/- 5% max/mmHg, P < 0.05) compared with the Ad.beta-Gal in CHF rats. Ad.nNOS also improved the baroreflex gain for the control of HR and RSNA (1.8 +/- 0.2 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 2.6 +/- 0.2 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01). In sham-operated rats, we found that Ad.nNOS transfer enhanced the HR range compared with Ad.beta-Gal gene transfer (188 +/- 15 vs. 127 +/- 14 beats/min, P < 0.05) but did not alter any other parameter. This study represents the first demonstration of altered baroreflex function following increases in central nNOS in the CHF state. We conclude that delivery of Ad.nNOS into the RVLM improves baroreflex function in rats with CHF.     

Regulation of cardiac adrenomedullin-mRNA in different stages of experimental heart failure

Adrenomedullin (AM) is a peptide hormone with vasodilating and natriuretic properties. AM plasma concentrations are elevated in heart failure. Whether cardiac AM-mRNA synthesis is increased in heart failure is not known. We measured AM-mRNA/GAPDH-mRNA in all four heart chambers in compensated and overt heart failure in rats with two different sizes of aortocaval shunt. Left and right atrial AM-mRNA expressions were unchanged in both heart failure models. Similarly, left and right ventricular AM-mRNA expressions were unchanged in compensated heart failure. In overt heart failure, however, the AM-mRNA expression was significantly increased in the left ventricle (145+/-20 vs. 100+/-3% of control, p<0.05). The right ventricular AM-mRNA expression was significantly increased only in a subgroup of animals with pulmonary congestion (lung weight >2.0 g, 141+/-16 vs. 100+/-11% of control, p<0.05). Ventricular AM concentrations were elevated in both ventricles in overt heart failure. AM plasma concentrations were significantly higher in the subgroup with pulmonary congestion than in rats with compensated heart failure (496+/-95 vs. 143+/-7 pmol/l, p<0.01). These data indicate that ventricular AM-mRNA expression and AM concentrations were upregulated only in advanced stages of heart failure. However, the exact contribution of cardiac AM synthesis to the increased AM plasma levels remains to be established.     

Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischemia and reperfusion

Adrenomedullin (AM) has been shown to protect against cardiac remodeling. In this study, we investigated the potential role of AM in myocardial ischemia-reperfusion (I/R) injury through adenovirus-mediated gene delivery. One week after AM gene delivery, rats were subjected to 30-min coronary occlusion, followed by 2-h reperfusion. AM gene transfer significantly reduced the ratio of infarct size to ischemic area at risk and the occurrence of sustained ventricular fibrillation compared with control rats. AM gene delivery also attenuated apoptosis, assessed by both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and DNA laddering. The effect of AM gene transfer on infarct size, arrhythmia, and apoptosis was abolished by an AM antagonist, calcitonin gene-related peptide [CGRP(8-37)]. Expression of human AM significantly increased cardiac cGMP levels and reduced superoxide production, superoxide density, NAD(P)H oxidase activity, p38 MAPK activation, and Bax levels. Moreover, AM increased Akt and Bad phosphorylation and Bcl-2 levels, but decreased caspase-3 activation. These results indicate that AM protects against myocardial infarction, arrhythmia, and apoptosis in I/R injury via suppression of oxidative stress-induced Bax and p38 MAPK phosphorylation and activation of the Akt-Bad-Bcl-2 signaling pathway. Successful application of this technology may have a protective effect in coronary artery diseases.     

Acute effects of methoxamine on left ventricular-arterial coupling in streptozotocin-diabetic rats: a pressure-volume analysis

We determined the acute effects of methoxamine, a specific alpha1-selective adrenoceptor agonist, on the left ventricular-arterial coupling in streptozotocin (STZ)-diabetic rats, using the end-systolic pressure-stroke volume relationships. Rats given STZ 65 mg x kg(-1) iv (n = 8) were compared with untreated age-matched controls (n = 8). A high-fidelity pressure sensor and an electromagnetic flow probe measured left ventricular (LV) pressure and ascending aortic flow, respectively. Both LV end-systolic elastance E(LV,ES) and effective arterial elastance Ea were estimated from the pressure-ejected volume loop. The optimal afterload Q(load) determined by the ratio of Ea to E(LV,ES) was used to measure the optimality of energy transmission from the left ventricle to the arterial system. In comparison with controls, diabetic rats had decreased LV end-systolic elastance E(LV,ES), at 513 +/- 30 vs. 613 +/- 29 mmHg x mL(-1), decreased effective arterial elastance Ea, at 296 +/- 20 vs. 572 +/- 48 mmHg x mL(-1), and decreased optimal afterload Q(load), at 0.938 +/- 0.007 vs. 0.985 +/- 0.009. Methoxamine administration to STZ-diabetic rats significantly increased LV end-systolic elastance E(LV,ES), from 513 +/- 30 to 602 +/- 38 mmHg x mL(-1), and effective arterial elastance Ea, from 296 +/- 20 to 371 +/- 28 mmHg x mL(-1), but did not change optimal afterload Q(load). We conclude that diabetes worsens not only the contractile function of the left ventricle, but also the matching condition for the left ventricular-arterial coupling. In STZ-diabetic rats, administration of methoxamine improves the contractile status of the ventricle and arteries, but not the optimality of energy transmission from the left ventricle to the arterial system.     

Effects of dietary phytoestrogens on cardiac remodeling secondary to chronic volume overload in female rats.

Previously, we demonstrated that intact female rats fed a standard rodent diet containing soybean products exhibit essentially no adverse left ventricular (LV) remodeling in response to aortocaval fistula-induced chronic volume overload. We hypothesized that phytoestrogenic compounds in the diet contributed to the female cardioprotection. To test this hypothesis, four groups of female rats were studied: sham-operated (Sham) and fistula (Fist) rats fed a diet with [P(+)] or without [P(-)] phytoestrogens. Eight weeks postfistula, systolic and diastolic cardiac function was assessed by using a blood-perfused, isolated heart preparation. High-phytoestrogen diet had no effect on body, heart, and lung weights, or cardiac function in Sham rats. Fistula groups developed LV hypertrophy, which was not reduced by dietary phytoestrogens [1,184 +/- 229 mg Fist-P(-) and 1,079 +/- 199 mg Fist-P(+) vs. 620 +/- 47 mg for combined Sham groups, P < 0.05]. Unstressed LV volume increased in Fist-P(-) rats (428 +/- 16 vs. 300 +/- 14 microl Sham, P < 0.0001), but it was not different from Sham for Fist-P(+) animals (286 +/- 17 microl). Fist-P(-) rats developed increased ventricular compliance (5.3 +/- 0.8 vs. 2.3 +/- 0.3 microl/mmHg Sham, P < 0.01), whereas Fist-P(+) rats had no change in compliance (2.8 +/- 0.4 mul/mmHg). Intrinsic ventricular contractility was maintained in the Fist-P(+) rats, but it was reduced (P < 0.001) in the Fist-P(-) rats [systolic pressure-volume slope: 1.04 +/- 0.03, 0.60 +/- 0.06, and 0.99 +/- 0.08 mmHg/microl, for Fist-P(+), Fist-P(-), and Sham, respectively]. These data indicate that dietary phytoestrogens contribute significantly to female cardioprotection against volume overload-induced adverse ventricular remodeling and that studies evaluating gender differences in cardiovascular remodeling must consider the influence of dietary phytoestrogens.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Decreased left ventricular function, myocarditis, and coronary arteriolar medial thickening following monocrotaline administration in adult rats.

Decreased right as well as left ventricular function can be associated with pulmonary hypertension (PH). Numerous investigations have examined cardiac function following induction of pulmonary hypertension with monocrotaline (MCT) assuming that MCT has no direct cardiac effect. We tested this assumption by examining left ventricular function and histology of isolated and perfused hearts from MCT-treated rats. Experiments were performed on 50 male Sprague-Dawley rats [348 +/- 6 g (SD)]. Thirty-seven rats received MCT (50 mg/kg sc; MCT group) while the remainder did not (Control group). Three weeks later, pulmonary artery pressure was assessed echocardiographically in 20 MCT and 8 Control rats. The hearts were then excised and perfused in the constant pressure Langendorff mode to determine peak left ventricular pressure (LVP), the peak instantaneous rate of pressure increase (+dP/dtmax) and decrease (-dP/dtmax), as well as the rate pressure product (RPP). Histological sections were subsequently examined. Pulmonary artery pressure was higher in the MCT-treated group compared with the Control group [12.9 +/- 6 vs. 51 +/- 35.3 mmHg (P < 0.01)]. Left ventricular systolic function and diastolic relaxation were decreased in the MCT group compared with the Control group (+dP/dtmax 4,178 +/- 388 vs. 2,801 +/- 503 mmHg/s, LVP 115 +/- 11 vs. 83 +/- 14 mmHg, RPP 33,688 +/- 1,910 vs. 23,541 +/- 3,858 beats x min(-1) x mmHg(-1), -dP/dtmax -3,036 +/- 247 vs. -2,091 +/- 389 mmHg/s; P < 0.0001). The impairment of cardiac function was associated with myocarditis and coronary arteriolar medial thickening. Similarly depressed ventricular function and inflammatory infiltration was seen in 12 rats 7 days after MCT administration. Our findings appear unrelated to the degree of PH and indicate a direct cardiotoxic effect of MCT.     

Cardiac response to submaximal exercise in dogs susceptible to sudden cardiac death

The hemodynamic response to submaximal exercise was investigated in 38 mongrel dogs with healed anterior wall myocardial infarctions. The dogs were chronically instrumented to measure heart rate (HR), left ventricular pressure (LVP), LVP rate of change, and coronary blood flow. A 2 min coronary occlusion was initiated during the last minute of an exercise stress test and continued for 1 min after cessation of exercise. Nineteen dogs had ventricular fibrillation (susceptible) while 19 animals did not (resistant) during this test. The cardiac response to submaximal exercise was markedly different between the two groups. The susceptible dogs exhibited a significantly higher HR and left ventricular end-diastolic pressure (LVEDP) but a significantly lower left ventricular systolic pressure (LVSP) in response to exercise than did the resistant animals. (For example, response to 6.4 kph at 8% grade; HR, susceptible 201.4 +/- 5.1 beats/min vs. resistant 176.2 +/- 5.6 beats/min; LVEDP, susceptible 19.4 +/- 1.1 mmHg vs. resistant 12.3 +/- 1.7 mmHg; LVSP, susceptible 136.9 +/- 7.9 mmHg vs. resistant 154.6 +/- 9.8 mmHg.) beta-Adrenergic receptor blockade with propranolol reduced the difference noted in the HR response but exacerbated the LVP differences (response to 6.4 kph at 8% grade; HR, susceptible 163.4 +/- 4.7 mmHg vs. resistant 150.3 +/- 6.4 mmHg; LVEDP susceptible 28.4 +/- 2.1 mmHg vs. resistant 19.6 +/- 3.0 mmHg; LVSP, susceptible 122.2 +/- 8.1 mmHg vs. resistant 142.8 +/- 10.7 mmHg). These data indicate that the animals particularly vulnerable to ventricular fibrillation also exhibit a greater degree of left ventricular dysfunction and an increased sympathetic efferent activity.     

Repeated inhalation of adrenomedullin ameliorates pulmonary hypertension and survival in monocrotaline rats

Adrenomedullin (AM) is a potent vasodilator peptide. We investigated whether inhalation of aerosolized AM ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Male Wistar rats given MCT (MCT rats) were assigned to receive repeated inhalation of AM (n = 8) or 0.9% saline (n = 8). AM (5 mug/kg) or saline was inhaled as an aerosol using an ultrasonic nebulizer for 30 min four times a day. After 3 wk of inhalation therapy, mean pulmonary arterial pressure and total pulmonary resistance were markedly lower in rats treated with AM than in those given saline [mean pulmonary arterial pressure: 22 +/- 2 vs. 35 +/- 1 mmHg (-37%); total pulmonary resistance: 0.048 +/- 0.004 vs. 0.104 +/- 0.006 mmHg.ml(-1).min(-1).kg(-1) (-54%), both P < 0.01]. Neither systemic arterial pressure nor heart rate was altered. Inhalation of AM significantly attenuated the increase in medial wall thickness of peripheral pulmonary arteries in MCT rats. Kaplan-Meier survival curves demonstrated that MCT rats treated with aerosolized AM had a significantly higher survival rate than those given saline (70% vs. 10% 6-wk survival, log-rank test, P < 0.01). In conclusion, repeated inhalation of AM inhibited MCT-induced pulmonary hypertension without systemic hypotension and thereby improved survival in MCT rats.     

Effects of endothelin on adrenomedullin secretion and expression of adrenomedullin receptors in rat cardiomyocytes

Both endothelin (ET) and adrenomedullin (AM), produced by cardiac myocytes, are thought to be locally-acting hormones in the heart. Recently, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have been shown to function together to serve as AM receptors stimulating cAMP production. In the present study, we examined the effects of ET on AM secretion, intracellular cAMP response to AM, and gene expressions of CRLR and RAMPs in cultured cardiac myocytes. Synthetic ET-1 dose-dependently increased AM secretion from the cardiomyocytes. AM increased the intracellular cAMP level in a dose-dependent manner and the cAMP accumulation by AM was significantly amplified by 24 h preincubation with ET-1. 10 nmol/L ET-1 significantly increased the CRLR mRNA level without any effect on RAMP1 mRNA. 1 micromol/L ET-1 significantly reduced the RAMP2 mRNA level, but ET-1 dose-dependently increased the RAMP3 mRNA level in the cardiac myocytes. These findings suggest that ET-1 not only stimulates AM secretion, but also modulates intracellular cAMP responses to AM probably by altering the expressions of CRLR and RAMPs in rat cardiomyocytes.     

Blocking the MyD88-dependent pathway protects the myocardium from ischemia/reperfusion injury in rat hearts

We examined whether blocking the MyD88 mediated pathway could protect myocardium from ischemia/reperfusion (I/R) injury by transfecting Ad5-dnMyD88 into the myocardium of rats (n=8) 3 days before the hearts were subjected to ischemia (45min) and reperfusion (4h). Ad5-GFP served as control (n=8). One group of rats was (n=8) subjected to I/R without transfection. Transfection of Ad5-dnMyD88 significantly reduced infarct size by 53.6% compared with the I/R group (15.1+/-3.02 vs 32.5+/-2.59) while transfection of Ad5-GFP did not affect I/R induced myocardial injury (35.4+/-2.59 vs 32.5+/-2.59). Transfection of Ad5-dnMyD88 significantly inhibited I/R-enhanced NFkappaB activity by 50% and increased the levels of phospho-Akt by 35.6% and BCL-2 by 81%, respectively. Cardiac myocyte apoptosis after I/R was significantly reduced by 59% in the Ad5-dnMyD88 group. The results demonstrate that both inhibition of the NFkappaB activation pathway and activation of the Akt signaling pathway may be responsible for the protective effect of transfection of dominant negative MyD88.     

Systolic elastance and resistance in the regulation of cardiac pumping function in early streptozotocin-diabetic rats

We determined the roles of maximal systolic elastance (E(max)) and theoretical maximum flow ((max)) in the regulation of cardiac pumping function in early streptozotocin (STZ)-diabetic rats. Physically, E(max) can reflect the intrinsic contractility of the myocardium as an intact heart, and (max) has an inverse relation to the systolic resistance of the left ventricle. Rats given STZ 65 mg/kg i.v. (n = 17) were divided into two groups, 1 week and 4 weeks after induction of diabetes, and compared with untreated age-matched controls (n = 15). Left ventricular (LV) pressure and ascending aortic flow signals were recorded to calculate E(max) and (max), using the elastance-resistance model. After 1 or 4 weeks, STZ-diabetic animals show an increase in effective LV end-diastolic volume (V(eed)), no significant change in peak isovolumic pressure (P(iso)(max)), and a decline in effective arterial volume elastance (E(a)). The maximal systolic elastance E(max) is reduced from 751.5 +/- 23.1 mmHg/ml in controls to 514.1 +/- 22.4 mmHg/ml in 1- and 538.4 +/- 33.8 mmHg/ml in 4-week diabetic rats. Since E(max) equals P(iso)(max)/V(eed), an increase in V(eed) with unaltered P(iso)(max) may primarily act to diminish E(max) so that the intrinsic contractility of the diabetic heart is impaired. By contrast, STZ-diabetic rats have higher theoretical maximum flow (max) (40.9 +/- 2.8 ml/s in 1- and 44.5 +/- 3.8 ml/s in 4-week diabetic rats) than do controls (30.7 +/- 1.7 ml/s). There exists an inverse relation between (max) and E(a) when a linear regression of (max) on E(a) is performed over all animals studied (r = 0.65, p < 0.01). The enhanced (max) is indicative of the decline in systolic resistance of the diabetic rat heart. The opposing effects of enhanced (max) and reduced E(max) may negate each other, and then the cardiac pumping function of the early STZ-diabetic rat heart could be preserved before cardiac failure occurs.     

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.     

The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation

In this work we aimed to observe (1) the changes in adrenomedullin (AM) and its receptor system - calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) - in myocardial ischemic injury and (2) the response of injuried myocardia to AM and the phosphorylation of Akt to illustrate the protective mechanism of AM in ischemic myocardia. Male SD rats were subcutaneously injected with isoproterenol (ISO) to induce myocardial ischemia. The mRNA levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 were determined by RT-PCR. Protein levels of Akt, phosphor-Akt, CRLR, RAMP1, RAMP2 and RAMP3 were assayed by Western blot. Results showed that, compared with that of the controls, ISO-treated rats showed lower cardiac function and myocardial injury. The mRNA relative amount of AM, CRLR, RAMP1, RAMP2 and RAMP3 in the myocardia of ISO-treated rats was increased. The elevated mRNA levels of CRLR, RAMP1, RAMP2 and RAMP3 were positively correlated with AM content in injured myocardia. The protein levels of CRLR, RAMP1, RAMP2 and RAMP3 in injured myocardia were increased compared with that of control myocardia. AM-stimulated cAMP generation in myocardia was elevated in the ISO group, and was antagonized by AM(22-52) and CGRP(8-37). Western blot analyses revealed that AM significantly enhanced Akt phosphorylation in injured myocardia, which was blocked by pretreatment with AM(22-52) or CGRP(8-37). Ischemia-injured myocardia hyper-expressed AM and its receptors - CRLR, RAMP1, RAMP2 and RAMP3 - and the response of ischemic myocardia to AM was potentiated, and the level of Akt phosphorylation was also increased, which suggests that changes in cardiac AM/AM receptor might play an important role in the pathogenesis of myocardial ischemic injury.     

Impact of nitric oxide on adrenomedullin- and proadrenomedullin N-terminal 20 peptide-induced cardiac responses: action by alone and combined administration

The cardiac effects of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) as well as the possible signaling pathways were investigated. In the isolated perfused rat heart, infusion of AM (10(-11) to 10(-8) M) and PAMP(10(-11) to 10(-8) M) for 10 min, alone or in combination, induced concentration-dependent decreases in the left ventricular pressure (LVP), LVP +/- dp/dtmax of the hearts. The effects were attenuated by Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase. ADM and PAMP alone or in combinations increased the coronary fluid (CF), which could be antagonized by L-NAME. Pretreatment of H89, an inhibitor of protein kinase A (PKA), failed to alter the AM- or PAMP-induced decreases in LVP and LVP +/- dp/dtmax, but further promoted the AM or PAMP increased CF. The cAMP content in left cardiac ventricle was increased significantly by ADM infusions but not by PAMP. There was no statistical difference in cAMP contents with ADM administrated alone from those combined with ADM and PAMP. In conclusion, this study reveals that ADM and PAMP infused alone or in combinations inhibited the function of rat hearts in vitro, which may be partly involved with the NOS/NO pathway, rather than cAMP/PKA.     

A review of the biological properties and clinical implications of adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating peptides.

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.     

Impaired parathyroid hormone action on urinary phosphorus excretion in isolated perfused kidney of streptozotocin-induced diabetic rats.

To study the effects of diabetes on the renal actions of parathyroid hormone (PTH), we observed urinary excretion of cyclic adenosine monophosphate (cAMP) and phosphorus in isolated perfused rat kidney. Diabetic rats were kept for 7 days after an intraperitoneal injection of 70 mg/kg streptozotocin (STZ). STZ-induced diabetic rats were treated with a daily injection of 20 U/kg lente-type insulin for 7 days. Plasma albumin, calcium, phosphorus, and PTH levels were not different among normal control, diabetic and insulin-treated diabetic groups. In the control rat kidney, the addition of PTH increased urinary cAMP excretion from 8 +/- 3 to 190 +/- 49 pmol/5 min and urinary phosphorus excretion from 11.3 +/- 4.4 to 33.6 +/- 10.8 microg/5 min. In the STZ-diabetic rat kidney, basal urinary cAMP was impaired, and PTH altered neither urinary cAMP nor phosphorus excretion (from below 0.7 to below 0.7 pmol/5 min, and from 15.5 +/-4.5 to 13.6 +/- 8.1 microg/5 min, respectively). Insulin treatment completely recovered the PTH actions. These results show that insulinopenic diabetes induces PTH resistance in the kidney.     

Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 +/- 0.3 mo (n = 9)] and old rats [30.6 +/- 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 +/- 790 vs. 11,106 +/- 555 mmHg/s, P < 0.05) and isovolumic relaxation time (tau) was increased (8.7 +/- 0.7 vs. 6.3 +/- 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after beta-adrenergic receptor stimulation with dobutamine (20 mug/kg), which increased LV dP/dt by 170 +/- 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 +/- 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 +/- 0.2 vs. 1.3 +/- 0.2%), alpha-tubulin (92%), and beta-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins.