To produce a prolonged decrease m 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 mflammallon or immune reaction AAV enters nondtvlchng cells and does not replicate Therefore, it IS an appropriate choice for gene therapy Recombinant AAV was prepared with a cassette contaumg a cytomegalovtrus promoter and the cDNA for the AT, receptor inserted m the annsense dire&on The cassette was packaged ohgonucleotides, directed to either AT,-R mRNA or to anglotensmogen mRNA, slgmfscantly reduce blood pressure m hypertensive animals with a single mJection mto the bram I-3 Although the adn-nmstration of antisense m the brain proved that antlsense can reduce high blood pressure of neurogemc orlgm, it obviously 1s not an acceptable route for treatment of human hypertension To demonstrate that antisense acts via a systemic route of delivery, we have shown that antisense delivered mtravenously4 or mtra-arterially5 can also reduce blood pressure m hypertensive rats. Antisense AT1 mRNA significantly decreased the blood pressure m 2&idney, 1 clip rats, m which circulating remn-Ang levels are high 4 Anglotensmogen mRNA-directed antIsense ohgonucleotide, in a hposome carrier inJected mtravenously m SHR, also decreased hypertension 5 The uptake of antisense was predommantly m the hver, as shown by fluorescent-tagged antisense. A sm-nlar approach was taken by Tomlta et al," who prepared three angiotensmogen mRNA-directed antisense ohgonucleotides and delivered them m hposomes and Sendal virus by direct mJectlon mto the hepatlc portal vem They also noted a decrease m blood pressure m SHR While these results have been encouraging for the use of antisense as a poFrom the Department of Physiology, College of Medicine, Unlversity of Florlda, Gamesvllle, Fla, and Harvard Medical School (P W ), Boston, MassCorrespondence to Dr M I Pixlhps, Department of Physiology, College of Medicine, Utuverslty of Flonda, Gamesvllle, FL 32610 E-mad MIP@phys med ufl edu 0 1997 American Heart Assoclatron, Inc tential treatment of hypertension, the maximum effectlveness of a single injection lasts for 7 days. Although this 1s Impressively longer than the response to a single dose of any antlhypertenslve drug currently available, it 1s our hope that we can extend the effectiveness of the antisense approach by delivering antisense m a viral vector that will produce a prolonged reduction m blood pressure for weeks or months There are several vu-al vectors to choose from, mcludmg retrovlruses, adenovlrus, herpes virus, polo virus, and AAV All have disadvantages and some advantages, but the AAV offers the most attractive advantages and the fewest disadvantages AAV 1s safe to use It does not induce any pathogenic response and does not replicate mslde cells The AAV 1s a defective parvovlrus and cannot replicate m cells without the presence of wild-type adenovlrus 73 The AAV IS effective as a vector because it either mt...
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.
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.
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.
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