Stan S. Greenberg

Rapid Induction of Messenger RNA for Nitric Oxide Synthase II in Rat Neutrophils In Vivo by Endotoxin and its Suppression by Prednisolone

Abstract Nitric oxide is believed to participate in nonspecific cellular immunity. Gram negative bacterial endotoxins increase the production of reactive nitrogen intermediates (RNI) in phagocytic cells by inducing the enzyme nitric oxide synthase II (NOSII). Anti-inflammatory glucocorticoids attenuate endotoxin-induced increases in RNI. This study evaluated the effect of in vivo administration of prednisolone on Escherichia coli lipopolysaccharide endotoxin (LPS)-induced increases in plasma RNI and neutrophil mRNA for NOS II and production of RNI in the rat. We show that LPS rapidly induces mRNA for NOS II and production of RNI (NO2- and NO3- anion) in rat neutrophils within 2 hr after in vivo administration of a sublethal dose of 0.5 mg/kg, iv. A pharmacologic dose of prednisolone (50 μg/kg, im) given 15 min before LPS-attenuated production of NO2- and NO3- by neutrophils and suppressed LPS-stimulated mRNA for NOS II. 3-Amino, 1,2,4-triazine inhibited NO2- and NO3- production without affecting gene expression for NOS II. These data demonstrate that LPS rapidly induces functional gene expression for NOS II and prednisolone prevents induction of NOS II activity by inhibiting transcription of its mRNA.

Changes in protein kinase C in early cardiomyopathy and in gracilis muscle in the BB/Wor diabetic rat

Hyperglycemia can upregulate protein kinase C (PKC), which may be an important mediator of the progression from normal heart and muscle function to diabetic myopathy in the myocardium and skeletal muscle in type 1 insulin-dependent diabetes mellitus (IDM). We evaluated this possibility during the early stage of IDM in BB/Wor diabetic (D) rats and age-matched BB/Wor diabetes-resistant (DR) rats. Interventricular septal thickness, E wave peak velocity of tricuspid inflow (both minimum and maximum), and left ventricular (LV) weight index were increased, and the rate of change in LV pressure (LV dP/dt) decreased in D rats subjected to M-mode and two-dimensional echocardiography and hemodynamic recording of heart rate, LV pressure (LVP), + LV dP/dt, -LV dP/dt, and LV end-diastolic pressure (LVEDP) in vivo and in vitro 41 days after the onset of hyperglycemia. Whole ventricle basal PKC activity was increased by 44.4 and 18.4% in the particulate and soluble fractions, respectively, from D rats compared with that from DR rats using r-32P phosphorylation of appropriate peptide substrates. When measured by Western blot gel densitometry, particulate PKC-alpha and PKC-delta content increased by 89 and 24%, respectively, but soluble PKC-beta and soluble and particulate PKC-epsilon were unchanged compared with that of DR rats. Similarly, gracilis muscle PKC activity and PKC-alpha and PKC-delta were elevated in the gracilis muscle, whereas that of the circulating neutrophil did not differ between the D and DR rats. Thus, in vivo, the early diabetic cardiomyopathy of the D rat is characterized by a restrictive LV with increased septal thickness and is associated with elevated PKC activity and increased amounts of myocardial particulate PKC-alpha and PKC-delta, which are also seen in the skeletal muscle. We conclude that increased PKC isozymes may play a pivotal role during IDM in the development of diabetic cardiomyopathy and skeletal muscle myopathy.Hyperglycemia can upregulate protein kinase C (PKC), which may be an important mediator of the progression from normal heart and muscle function to diabetic myopathy in the myocardium and skeletal muscle in type 1 insulin-dependent diabetes mellitus (IDM). We evaluated this possibility during the early stage of IDM in BB/Wor diabetic (D) rats and age-matched BB/Wor diabetes-resistant (DR) rats. Interventricular septal thickness, E wave peak velocity of tricuspid inflow (both minimum and maximum), and left ventricular (LV) weight index were increased, and the rate of change in LV pressure (LV dP/d t) decreased in D rats subjected to M-mode and two-dimensional echocardiography and hemodynamic recording of heart rate, LV pressure (LVP), +LV dP/d t, -LV dP/d t, and LV end-diastolic pressure (LVEDP) in vivo and in vitro 41 days after the onset of hyperglycemia. Whole ventricle basal PKC activity was increased by 44.4 and 18.4% in the particulate and soluble fractions, respectively, from D rats compared with that from DR rats using r-32P phosphorylation of appropriate peptide substrates. When measured by Western blot gel densitometry, particulate PKC-α and PKC-δ content increased by 89 and 24%, respectively, but soluble PKC-β and soluble and particulate PKC-ε were unchanged compared with that of DR rats. Similarly, gracilis muscle PKC activity and PKC-α and PKC-δ were elevated in the gracilis muscle, whereas that of the circulating neutrophil did not differ between the D and DR rats. Thus, in vivo, the early diabetic cardiomyopathy of the D rat is characterized by a restrictive LV with increased septal thickness and is associated with elevated PKC activity and increased amounts of myocardial particulate PKC-α and PKC-δ, which are also seen in the skeletal muscle. We conclude that increased PKC isozymes may play a pivotal role during IDM in the development of diabetic cardiomyopathy and skeletal muscle myopathy.

Ouabain Enhances Basal Release of Nitric Oxide from Carotid Artery

The authors tested the postulate that ouabain releases nitric oxide (NO) from the vascular endothelium of porcine carotid arteries (PCAs) with the technique of perfusion-superfusion bioassay, in which the perfused PCA with endothelium served as the source of NO and superfused left circumflex coronary artery (CMFX) rings with rubbed endothelium served as the bioassay tissue. Selective exposure of the PCA to ouabain (10 microM) enhanced the basal release of NO but did not affect bradykinin-stimulated (BK; 0.1-100 picomoles) release of NO. The effect of ouabain on basal release of NO from PCA persisted after pretreatment of either PCA or circumflex coronary artery with propranolol (1 microM); ibuprofen (1 microM); and hydrocortisone (10 microM). Finally, selective pretreatment of the PCA with L-NG-monomethylarginine (LNMMA; 100 microM) to inhibit 1-arginine-derived NO synthesis inhibited the relaxation of the circumflex coronary artery to basal, BK, and ouabain-stimulated effluent. Since a nonspecific increase in intracellular calcium ion will enhance both basal and agonist-induced release of NO, the authors conclude that a ouabain-sensitive ATPase is involved in basal release of NO from the endothelium of the PCA. Alternatively, ouabain may act on an isozyme of NO synthase in the vascular endothelium. Speculatively, ouabain-induced stimulation of NO release from vascular endothelium may contribute to the beneficial effect of ouabain in congestive heart failure.

Rapid Induction of mRNA for Nitric Oxide Synthase II in Rat Alveolar Macrophages by Intratracheal Administration of Mycobacterium Tuberculosis and Mycobacterium Avium

Abstract Mycobacterium avium complex (MAC) organisms are among the most common bacterial cause of disseminated infection in patients with acquired immune deficiency syndrome (AIDS). An increase in the incidence of virulent Mycobacterium tuberculosis (MTB) is also occurring throughout the world. In vitro data suggest that nitric oxide (NO) may be important in restricting the growth of MAC. However, the ability of MTB to stimulate NO production and the susceptibility of MTB to the bactericidal activity of NO produced by murine alveolar macrophages (AM) is controversial. This study tested the hypothesis that in vivo administration of heat-killed MAC (strain 100 and 101) and human virulent MTB (strain F1) to rats stimulated NO production by rat AM, ex vivo. We show that heat-killed MTB instilled into rat lungs rapidly induced mRNA for NO synthase (iNOS) II in AM obtained by bronchoalveolar lavage (BAL). In contrast, expression of AM iNOS mRNA was only found in 40% of the rats given MAC. Moreover, the change in iNOS mRNA in the AM obtained from rats given MTB and MAC correlated with the production of the reactive nitrogen intermediates (RNI) NO2- and NO3- in BAL fluid, lung homogenate, and the spontaneous generation of RNI by isolated AM ex vivo and occurred without measurable increases in BAL fluid tumor necrosis factor-α (TNF-α). L-NG-monomethylarginine (50 mg/kg, ip) given 30 min before MAC or MTB attenuated the increase in RNI in lung homogenates and BAL fluid. This is the first demonstration that in vivo exposure to MTB results in rapid upregulation of gene expression for iNOS which is associated with functional RNI production by rat AM. These results show that MTB human virulent strain 1 has the ability to rapidly upregulate iNOS mRNA in AM. If human AM generate NO from L-arginine by either iNOS or other NADPH oxidases then NO may play a role in the overall host-defense response of the lung to MAC and MTB.

In Vivo Administration of Endotoxin and Tumor Necrosis Factor-α Produce Different Effects on Constitutive and Inducible Nitric Oxide Synthase Activity in Rat Neutrophils and Aorta Ex Vivo

Abstract Tumor necrosis factor-α (TNF-α) inhibits release of nitric oxide (NO) in vitro by stimulating the degradation of constitutive NO synthase (cNOS III) mRNA. However, TNF-α is believed to be the cytokine mediator of the hypotension and upregulation of inducible NO synthase (iNOS II) produced by gram-negative bacterial endotoxin (LPS). Some in vivo effects of TNF-α are opposite to those which occur in vitro. This study tested the hypothesis that in vivo administration of exogenous TNF-α and endogenously released TNF-α induce iNOS II activity and inhibit cNOS III activity, and thereby mediate the acute phase effects of LPS on blood pressure and the NO system in the rat. We show that LPS produces acute phase hypotension in ketamine anesthetized rats. The hypotension was associated with elevation of biologically active TNF-α in plasma, increased production of RNI (NO2 - and NO3 - anion) in rat neutrophils (PMN) and suppression of RNI production by A23187 (1 μM) stimulated thoracic aorta (RTA) ex vivo. TNA-α (106 U/ml, iv) did not produce acute phase hypotension but initially raised arterial blood pressure and heart rate (HR), did not increase RNI production by PMN, and inhibited RNI production by A23187 stimulated RTA ex vivo. Pretreatment of rats with the Immunex monomeric soluble P75 receptor binding protein for TNF-α (TNFsr, 0.5 mg/kg, iv) 15 min prior to LPS administration decreased circulating TNF-α from 92,137 ± 12,456 U/ml to undetectable levels as determined by the L929 bioassay. However, LPS-induced increases in RNI in PMN was enhanced and LPS-induced decreases in RNI production by RTA was inhibited by TNFsr. Thus, in vivo administration of TNF-α does not mimic the hemodynamic and NO-inducing effects of LPS. However, TNF-α mediates in part LPS-induced inhibition of RNI production by RTA.

Tumor Necrosis Factorα Inhibits Contractions to Sympathetic Nerve Stimulation by a Nitric Oxide-Dependent Mechanism

Summary Abstract. Gram-negative sepsis and administration of tumor necrosis factorα (TNFα) are associated with hypotension and peripheral neuropathies suggestive of impaired sympathetic neurotransmission. We examined the effect of TNFα on the responses of the bovine pulmonary artery (BPA) to transmural sympathetic nerve stimulation (SNS). BPA contracted to SNS (0.5-32 Hz, 5-10 V, 2-msec duration, 2-msec delay) in a frequency-dependent manner. The contractions of the BPA to SNS were mediated by norepinephrine and activation of postsynaptic α1-adrenoceptors, since they were attenuated by prazosin. Maximum contraction of the BPA to SNS was significantly enhanced (148 ± 37% increase, n = 6) after inhibition of nitric oxide synthase with L-NG-monomethylarginine (LNMMA, 500 μM), an effect abrogated by L-arginine (1 mM). TNFα (0.0042, 0.042, and 0.42 μg/ml) selectively inhibited contractions of the BPA to SNS without affecting the contraction of the BPA to exogenous norepinephrine. In BPA incubated with LNMMA (5-500 μM), TNFα facilitated rather than inhibited SNS. TNFα increased the formation of amperiometrically measured free nitric oxide in bovine adrenal chromaffin cells in primary culture. The data show that in the absence of LNMMA, TNFα releases free nitric oxide from a sympathetic neuron and selectively inhibits the contractions of the BPA to SNS. In BPA in which nitric oxide synthase I is inhibited by LNMMA, TNFα amplifies the contractions to SNS, even in the absence of endothelium. Thus, TNFα can modify vascular smooth muscle tone by affecting SNS. TNFα inhibits SNS at the level of the neuron by a mechanism involving the L-arginine-nitric oxide pathway. TNFα-induced suppression of SNS and neurotransmission may contribute to the hypotension and peripheral neuropathy of sepsis.

PROTEIN KINASE C ISOZYMES IN SKELETAL MUSCLES DURING THE EARLY STAGE OF GENETIC AND STREPTOZOCIN DIABETES

Abstract This study examined the changes in PKC isozyme activity, content, and cellular distribution in rat gastrocnemius and soleus muscles prior to any evidence of neural degeneration or impaired skeletal muscle function, during the onset of streptozocin-induced (STZ) and genetic diabetes mellitus (DM). PKC activity was increased more in the particulate than in the soluble fractions of the soleus and gastrocnemius muscles obtained from rats treated with STZ and the gastrocnemius muscle obtained from BB-Wor diabetic rats (D rats). The predominant constitutive PKC isozymes in the skeletal muscles obtained from the STZ-treated and D rats were PKCα ≫ PKC∊ > PKCδ as determined by Western immunoblot assay. The content of each PKC isozyme did not differ between the soleus and gastrocnemius muscles of the control Sprague-Dawley rats for the STZ-treated rats and the BB Wor diabetic resistant (DR) rats. Moreover, the PKC isozyme content did not differ in the soluble fraction of D or STZ rats when compared to their corresponding control animals. PKCδ increased more than PKCα or PKC∊ in the particulate fraction of gastrocnemius and soleus muscles when obtained from either D or STZ rats. Since similar changes in skeletal muscle PKC isozyme profiles occurred independent of the duration of the diabetes and thereby the degree of nerve degeneration, insulin resistance, and the model of DM tested, we conclude that changes in skeletal muscle PKC precede the skeletal muscle myopathy of DM.

Neural Nitric Oxide Synthase and Neutrophil Function

Nitric oxide synthases (NOS, EC 1.14.13.39) are members of a family of cytochrome P450-like reductases linked to an NADPH oxidase enzyme. Nitric oxide synthase converts L-arginine to nitric oxide (NO) and L-citrulline utilizing the electrons generated by the reduction of oxygen to superoxide, hydrogen peroxide, and ultimately water (Wang and Marsden,1995,Xie and Nathan,1994; Forstermann and Kleinert,1995). At least three isozymes of NOS exist, each of which is derived from a different gene (Forstermann and Kleinert,1995). Endothelial NOS (ecNOS) and neural NOS (nNOS) are constitutive, calcium-activated isozymes found in endothelial and endocardial cells, airway epithelial cells, adrenal cortical and medullary cells, neurons, skeletal muscle, and many soft tissues (Forstermann and Kleinert,1995;Xie and Nathan,1994;Forstermann and Kleinert,1995). Inducible NOS (iNOS), primarily regulated by transcription, is elicited in inflammatory and immunocompetent cells; cardiac, striated, and smooth muscles; pancreatic beta cells; and a wide variety of tissues by bacterial endotoxins, cytokines, and autacoids (Wang and Marsden,1995; Xie and Nathan, 1994;Forstermann and Kleinert,1995;Greenberg et al.,1996,Greenberg et al.,1997;Nichols et al.,1988;Kwon et al.,1996).