Phorbol ester stimulates the synthesis and secretion of brain natriuretic peptide from neonatal rat ventricular cardiocytes: a comparison with the regulation of atrial natriuretic factor.

During left ventricular hypertrophy, brain natriuretic peptide (BNP) and atrial natriuretic factor (ANF) mRNA levels increase, possibly due to stretch-induced activation of protein kinase C. Phorbol ester treatment of primary cultures of neonatal rat ventricular cardiocytes represents an in vitro model of hypertrophic cell growth and has previously been shown to stimulate ANF synthesis and secretion. Using this model, we studied the synthesis and secretion of BNP to determine whether its regulation in cardiac cells is similar to ANF. Addition of 10(-7) M phorbol 12-myristate 13-acetate (PMA) resulted in a 3- to 4-fold increase in immunoreactive BNP (irBNP) secretion 24-48 h after treatment. Over a concentration range of 10(-8)-10(-6) M, PMA increased irBNP secretion to equivalent levels. Another phorbol ester agonist, phorbol 12,13-didecanoate, stimulated irBNP secretion, while the inactive analog 4 alpha-phorbol 12,13-didecanoate had no effect. Inhibition of protein kinase C (PKC) with 10(-8) M staurosporine decreased basal secretion of irBNP 60% and prevented PMA induction of irBNP, whereas both stimulated and basal secretion of ANF were minimally affected. BNP mRNA increased 6-fold by 3 h of PMA treatment and remained elevated above control levels for 48 h. Staurosporine prevented the increase in BNP mRNA. To determine whether PKC or a PKC-dependent pathway was involved in persistent stimulation of BNP and ANF in cells chronically treated with PMA, ventricular cardiocytes were treated with PMA for 24 h, followed by PMA plus 10(-8) M staurosporine for 24 h. BNP mRNA was reduced to control levels, while ANF mRNA was reduced by an average of 20%. To test whether mRNA stability was involved in the differential effect of chronic phorbol ester treatment, cardiocytes were treated with the protein synthesis inhibitor cycloheximide (20 micrograms/ml). BNP mRNA levels were stimulated as early as 30 min after treatment, but ANF mRNA remained unaffected. Cycloheximide also potentiated PMA's effect on BNP mRNA after 1.5, 9.5, and 24 h of treatment. To test whether a transcriptional mechanism was involved in the stimulation of BNP mRNA by PMA, cells were treated with the inhibitor actinomycin D (5 micrograms/ml) for 24 h in the presence of PMA. Actinomycin D reduced the stimulatory effect of PMA on BNP mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Phospholipase A2 metabolites regulate inducible nitric oxide synthase in myocytes.

The proinflammatory cytokine interleukin-1beta (IL) stimulates inducible nitric oxide synthase (iNOS) mRNA, protein, and nitric oxide (NO) production in neonatal ventricular myocytes (NVM). In other types of cells, IL also activates phospholipase A2 (PLA2), which liberates arachidonic acid for the pathways involved in eicosanoid production, and induces the cyclooxygenase-2 (COX-2) isoform, which increases prostanoid production. Since NO has been shown to directly stimulate COX activity and the resulting prostanoids to modulate IL induction of iNOS, we questioned whether PLA2 and/or COX products are involved in IL regulation of iNOS and NO production in NVM. We first found that IL induced COX-2 mRNA and protein, resulting in approximately 200-fold and 15-fold increases in PGE2 and 6-keto-PGF1alpha (the stable metabolite of PGI2), respectively. IL-stimulated prostanoid production was inhibited by the COX-2-specific inhibitor NS-398, as well as the nonspecific COX inhibitor indomethacin (INDO). We next studied the involvement of the PLA2 inhibitor ONO-RS-082 (ONO) and the COX inhibitor INDO in IL regulation of iNOS. Pretreatment with ONO blocked IL-stimulated NO production and iNOS protein, suggesting that PLA2 products are involved in regulation of iNOS synthesis. Unlike ONO, the COX inhibitor INDO had little effect on IL-stimulated NO. In addition to the COX pathway, arachidonic acid (AA) is also metabolized by the lipoxygenase (LO) pathway. The LO inhibitor nordihydroguaiaretic acid (NDGA) decreased IL-stimulated NO and iNOS synthesis. These data suggest that: (1) IL upregulates COX-2 expression and prostanoid production in NVM; and (2) AA metabolites other than COX products, possibly products of the LO pathway, are involved in IL regulation of iNOS.

Mechanisms of interleukin-1beta regulation of nitric oxide synthase in cardiac myocytes.

Cytokines and endotoxin stimulate inducible NO synthase (iNOS) in different types of cells; however, little is known about regulatory mechanisms. Using the Griess reagent for nitric levels, Western blots for iNOS protein, Northern blots for iNOS mRNA, and transient transfection studies to monitor transcription, we determined potential mechanisms involved in interleukin-1beta stimulation of iNOS in cultured neonatal ventricular myocytes. When myocytes were treated with interleukin-1beta (5 ng/mL), nitrite levels increased, and this effect was inhibited 80% by the specific iNOS inhibitor aminoguanidine. Neither interferon gamma nor tumor necrosis factor-alpha alone stimulated nitrite production. Bacterial endotoxin alone stimulated nitrites and potentiated the effect of interleukin. To determine whether a tyrosine kinase-mediated signaling pathway was involved in interleukin action, we used the inhibitor genistein, which blocked interleukin-stimulated nitrites, iNOS protein, and iNOS mRNA. To determine the effect of activation of protein kinase C, we treated cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA decreased both interleukin-stimulated nitrites and iNOS protein by 40%. To determine the involvement of cyclic nucleotides, cells were treated with either dibutyryl cAMP or cGMP. cAMP (1 mmol/L) stimulated iNOS mRNA, protein, and nitrite production, whereas cGMP had no effect. To test for a direct effect of interleukin on transcription of the iNOS gene, we transfected the full-length mouse iNOS 5' regulatory sequences (-1592 to +160) coupled to a luciferase reporter gene (-1592iNOSLuc). Interleukin stimulated luciferase activity 1.8 +/- 0.2-fold. To determine whether interleukin also affects iNOS mRNA stability, interleukin-stimulated iNOS mRNA was allowed to decay in the presence of the transcription inhibitor actinomycin D. iNOS mRNA t1/2 (approximately 1 hour) was not affected by interleukin. Thus, our data suggest that (1) interleukin-1beta is the primary cytokine in myocyte iNOS regulation and acts predominantly at the transcriptional level; (2) interleukin stimulation of iNOS mRNA and protein is coupled to a tyrosine kinase-mediated signaling pathway; and (3) protein kinase C and cAMP can modify interleukin signaling by decreasing and increasing iNOS, respectively.

Genotypic drug resistance and cause of death in HIV-infected persons who died in 1999.

We analyzed the relationship between viral drug resistance and causes of death in 29 HIV-1-infected patients who had been followed in an HIV-outpatient clinic and died in 1999. Six patients (21%) died with plasma HIV-RNA levels <1000 copies/ml. Seven (24%) died with wild-type (WT) virus in plasma, 6 (21%) had reverse transcriptase (RT) mutations only, 10 (34%) had multidrug-resistant (MDR) virus. The causes of death were not differently distributed among these groups; however, 8 of 16 patients (50%) with resistant viruses died of end-organ failure versus 2 of 7 patients (29%) with WT virus. Seventeen of 32 patients (53%) were thought by their physicians to be noncompliant with prescribed therapy. Major resistance mutations to antiretroviral drugs were present in viruses from at least 55% of our HIV-1-infected patients who died in 1999. Nonetheless, deaths also occurred among patients with well-controlled HIV infection and among patients with WT virus in plasma. Infections related to incomplete immune restoration, inability to maintain suppressive antiretroviral drug levels, and end-organ failures all contribute to mortalities in the era of highly active antiretroviral therapy.