Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of AT1-R mRNA antisense.

To produce a prolonged decrease in blood pressure, we have developed a nonpathogenic adeno-associated viral vector (AAV) with the antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV does not stimulate inflammation or immune reaction. AAV enters nondividing cells and does not replicate. Therefore, it is an appropriate choice for gene therapy. Recombinant AAV was prepared with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1 receptor inserted in the antisense direction. The cassette was packaged in the virion. Stable transfection of NG108-15 cells with the PAAV-AS (plasmid AAV) antisense to AT1-R produced a significant reduction in AT1 receptors. A single injection of the rAAV-AS (viral vector) was made in adult spontaneously hypertensive rats, either directly in the hypothalamus (1 microL) or in the lateral ventricles (5 microL). The result shows that there is a significant decrease of blood pressure (approximately 23 +/- 2 mm Hg) for up to 9 weeks after injection. Control injections of mock vector produced no change in blood pressure during the same time period in age-matched controls. In young spontaneously hypertensive rats (3 weeks), a single intracardiac injection of recombinant rAAV-AS reduced blood pressure and slowed the development of hypertension compared with controls (P < .01). The results suggest that a prolonged reduction in high blood pressure can be achieved with AAV vectors delivering antisense to inhibit AT1 receptors with a single administration.

Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells.

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.

Alpha-2 adrenoceptors in medullary thick ascending limbs of the rabbit kidney.

Marked differences in the characteristics of alpha-2 adrenoceptors in the kidney of various species have been reported. In addition, there are functional differences in alpha-2 adrenoceptors in various nephron segments of the same species. In this study we have characterized alpha-2 adrenoceptors in the medullary thick ascending limb (MTAL) isolated from the rabbit kidney. The equilibrium binding of [3H]rauwolscine to MTAL homogenates was measured after incubation for 45 min at 25 degrees C in the absence and presence of 10 microM phentolamine. The specific binding of [3H]rauwolscine was saturable with a Kd of 2.6 +/- 0.1 nM and maximal binding of 234 +/- 18 fmol/mg of protein. Adrenergic drugs competed with MTAL-bound [3H]rauwolscine with the following order of potency: 1) antagonists: rauwolscine = yohimbine > phentolamine >> prazosin = propranolol; and 2) agonists: oxymetazoline > clonidine > epinephrine > BHT 933 (azepexole) > alpha-methylnorepinephrine. Our results indicate that specific alpha-2 adrenoceptors are present in the rabbit MTAL. The high (> 1600) ratio of Ki of prazosin: oxymetazoline suggests that the alpha-2 adrenoceptors in the rabbit MTAL belong to an alpha-2A subtype. Guanabenz, iodoclonidine and epinephrine at high concentrations decreased forskolin- and vasopressin-stimulated cyclic AMP formation in the rabbit MTAL. These results suggest that alpha-2 adrenoceptors are negatively coupled to adenylate cyclase system in the MTAL.

Muscarinic receptors in MDCK cells are coupled to multiple messenger systems.

Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.

Characterization of cholinergic receptors in Madin-Darby canine kidney cells.

Muscarinic-type cholinergic receptors coupled to the phosphoinositide (PI) second messenger system are reported to be present in the inner medullary collecting duct cells. Madin-Darby canine kidney (MDCK) cells have several characteristics of collecting duct cells and have been shown to respond to muscarinic agonists. To determine if MDCK cells have PI-coupled muscarinic receptors, the radioligand binding and the effects of cholinergic agonists and antagonists on PI hydrolysis in MDCK cells were studied. The specific binding of [3H]1-quinuclidinyl benzilate ([3H]QNB), a muscarinic antagonist, to MDCK cell membranes had a Kd = 88 +/- 7 pM and a Bmax = 1464 +/- 88 fmol/mg of protein. The displacement of [3H]QNB from MDCK cell membranes by various cholinergic antagonists and agonists showed the order of potency: atropine greater than 4-diphenylacetoxy N-methylpiperidine (4-DAMP) greater than p-fluorohexahydrosiladifenidol greater than pirenzepine greater than metoctramine greater than arecoline greater than carbachol. The cholinergic agonists carbachol and arecoline stimulated PI hydrolysis in a concentration-dependent manner with an EC50 of 3.7 and 1.3 microM, respectively. Muscarinic antagonists abolished carbachol-stimulated PI hydrolysis in the following order of potency: atropine greater than 4-DAMP greater than pirenzepine much greater than methoctramine. The order of potency of muscarinic antagonists is consistent with the characteristics of the M3 subtype of muscarinic receptors. It is concluded that: (1) muscarinic receptor density in MDCK cells is 50 times higher than that in inner medullary collecting duct cells; (2) muscarinic receptors in MDCK cells are putative M3 subtype; and (3) muscarinic receptors in MDCK cells are functionally coupled to the PI second messenger system. This intracellular messenger system may, at least, be partially responsible for the action of cholinergic agonists in these cells and in the kidney.

Significance of CoA-transferase in the reaction of maleate with amino acids and proteins.

Maleate reacts, in the presence of acetoacetyl-CoA and CoA-transferase with amino acids containing no sulphydryl groups. The physico-chemical properties of the reaction products are the same as those of the derivatives obtained in the reaction of amino acids with maleic anhydride, which indicates that the amino groups became acylated. In the presence of the transferase system, maleate also reacts with such proteins as histone IIA, cytochrome c and albumin. The reaction also occurs after blocking SH groups, thus it could involve amino groups as well. The reacting agent is not maleate itself but a product of the reaction of maleate with acetoacetyl-CoA, catalysed by the transferase, i.e. maleyl-CoA.