Evidence for a 12 kDa "Carrier Protein" for Natriuretic Hormone.

The search for the elusive Na-K-ATPase-inhibiting natriuretic hormone continues. In this review, evidence is presented that isolating the carrier protein for natriuretic hormone from hypertensive plasma is a necessary first step before splitting off the final hormone. The carrier protein has a molecular weight of 12 kDa while the final hormone has a molecular weight of 408 Da. Both compounds inhibit Na-K-ATPase but the compound containing the carrier protein predominates. The question has been raised as to whether the carrier protein is in actuality proANF, a 17 kDa protein that can be split between a 14 kDa protein (the presumptive proANF) and the 3 kDa ANF.

Lead-binding proteins: a review.

Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte). Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD) isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 µg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin ß4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

Peroxynitrite but not nitric oxide donors destroys epinephrine: HPLC measurement and rat aorta contractility.

Nitric oxide (NO) and peroxynitrite (ONOO) have been reported to destroy catecholamines. We compared the ability of NO donors and peroxynitrite to decompose epinephrine in both chemical and pharmacological experiments. Epinephrine (1 microM) was incubated with NO donors (SNAP and MAHMA NONOate) and ONOO at a concentration of 0.1 mM in phosphate buffer (pH 7.4; 0.1 M) or Krebs solution for 10 minutes at 37 degrees C. HPLC revealed that the concentration of epinephrine in the presence of NO donors was unaltered. In contrast, peroxynitrite decreased epinephrine concentration more than 20 fold. Similar relationships were obtained in the study of rat thoracic aorta ring contraction. The contractile activity (EC50) of epinephrine in control solutions and after incubation of NE with NO donors did not change. EC50 was measured at 8-10 nM in control solutions and after preincubation with NO donors. However when epinephrine was preincubated with peroxynitrite, no contractile effect was evoked. Therefore, under these experimental conditions peroxynitrite, but not NO donors, was capable of destroying epinephrine.

Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine.

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.

Interaction of dimercaptosuccinic acid (DMSA) with angiotensin II on calcium mobilization in vascular smooth muscle cells.

Dimercaptosuccinic acid (DMSA) was shown to lower blood pressure in rat models of arterial hypertension. Thus, there is evidence that-besides its chelating properties-DMSA has a direct vascular effect, e.g. through scavenging of reactive oxygen species (ROS). We speculated that, in addition, intracellular calcium mobilization may be involved in this action. Therefore, the present study examined the effects of DMSA on Ca(2+) mobilization in cultured vascular smooth muscle cells (VSMCs) from rat aorta. Intracellular free Ca(2+) concentration ([Ca(2+)](i)) was measured with fura-2 AM. In a first series of experiments DMSA, 10(-11) to 10(-6)M, induced an immediate dose-dependent up to 4-fold rise of [Ca(2+)](i) (P<0.001) which was almost completely blunted by the calcium channel blocker verapamil or the intracellular calcium release blocker TMB-8. In a second series of experiments, when VSMCs were exposed acutely to DMSA (10(-11) to 10(-6)M), the angiotensin (ANG) II (10(-8)M)-induced rise in [Ca(2+)](i) to 295+/-40nM was attenuated at the average by 49% independent of the dose of DMSA. Preincubation of VSMCs with DMSA (10(-6)M) for 60min reduced basal [Ca(2+)](i) by 77% (P<0.001) and dose-dependently attenuated the ANG II (10(-8)M)-induced rise in [Ca(2+)](i) between 28 and 69% at concentrations between 10(-9) and 10(-5)M DMSA, respectively (P<0.05 and <0.01). In the presence of TMB-8, which attenuated the ANG II (10(-8)M)-induced rise in [Ca(2+)](i) by 66%, DMSA (10(-6)M) had no additional suppressive effect on [Ca(2+)](i). The results suggest that DMSA acutely raises [Ca(2+)](i) by stimulating transmembrane calcium influx via L-type calcium channels and by calcium release from intracellular stores followed by a decrease in [Ca(2+)](i) probably due to cellular calcium depletion. Thus, in addition to its action as scavenger of ROS, which in part mediate the vasoconstrictor response, e.g. to ANG II, DMSA may exert its hypotensive effect through decreasing total cell calcium, thereby attenuating the vasoconstrictor-induced rise in [Ca(2+)](i) in VSMCs.