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These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats.

It has been demonstrated that gene transfer by in vivo electroporation of mouse muscle increases the level of gene expression by more than 100-fold over simple plasmid DNA injection. We tested continuous rat erythropoietin (Epo) delivery by this method in normal rats, using plasmid DNA expressing rat Epo (pCAGGS-Epo) as the vector. A pair of electrodes was inserted into the thigh muscles of rat hind limbs and 100 microg of pCAGGS-Epo was injected between the electrodes. Eight 100-V, 50-msec electric pulses were delivered through the electrodes. Each rat was injected with a total of 400 microg of pCAGGS-Epo, which was delivered to the medial and lateral sides of each thigh. The presence of vector-derived Epo mRNA at the DNA injection site was confirmed by RT-PCR. The serum Epo levels peaked at 122.2 +/- 33.0 mU/ml on day 7 and gradually decreased to 35.9 +/- 18.2 mU/ml on day 32. The hematocrit levels increased continuously, from the preinjection level of 49.5 +/- 1.1 to 67.8 +/- 2.2% on day 32 (p < 0.001). In pCAGGS-Epo treated rats, endogenous Epo secretion was downregulated on day 32. In a control experiment, intramuscular injection of pCAGGS-Epo without subsequent electroporation did not significantly enhance the serum Epo levels. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the continuous delivery of Epo.

The anemia associated with chronic renal failure is one of the best target diseases for erythropoietin (Epo) gene transfer. We previously reported a short-term (1 month) study of continuous rat Epo delivery by muscle-targeted gene transfer of plasmid DNA expressing rat Epo (pCAGGS-Epo) using in vivo electroporation in normal rats. Here, we performed a long-term pharmacokinetic study of continuous Epo delivery by this method in normal rats and uremic five-sixths nephrectomized rats. In normal rats, Epo gene expression and sufficient erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 11

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The pharmacological and biochemical mechanisms of contractile responses to the protein kinase C (PKC) activator phorbol-12,13-diacetate (PDA) were investigated in canine basilar arteries. In the normal medium, PDA elicited a strong, dose-related, and slow-developing sustained contraction. Among the constrictors examined, including serotonin, prostaglandin F2 alpha, and endothelin, only PDA yielded contractions in a Ca2(+)-free medium. In both media, the PDA-induced contractions were virtually inhibited by either staurosporine, H-7, or quinacrine, while neither neurotransmitter blockades nor R24571 (calmidazolium) exerted significant effects. In addition, it was shown that 8-bromocyclic GMP, but not 8-bromocyclic AMP, markedly curtailed the PDA-induced contractions. Biochemical analysis, furthermore, showed that PDA induced increased phosphorylations of 27- and 96-kDa and proteins other than the myosin light chain (MLC) 20-kDa protein. Thus, the present results open up a novel mechanism of sustained cerebral artery contractions, where PKC activation rather than Ca2+/calmodulin/MLC system plays a key role that is regulated both by phospholipase A2 and by cyclic GMP.

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.

We previously suggested that activation of the protein kinase C-mediated contractile system may participate in the occurrence of chronic cerebral vasospasm. In the present study, we compared segments of normal beagle basilar arteries in vitro with segments of arteries undergoing chronic vasospasm to determine the responsiveness to various agonists such as serotonin, prostaglandin F^, and phorbol 12,13-diacetate as well as to external Ca 2+. We also compared the effects of W-7 (a calmodulin inhibitor), nicardipine (a calcium channel blocker), and H-7 (a protein kinase C inhibitor) on the spontaneous tonus of arterial segments stabilized at a resting tension of 3 g. Compared with normal segments, the responsiveness to each agonist in segments undergoing vasospasm was essentially unchanged whereas the responsiveness to external Ca 2+ was significantly decreased (/><0.001). In segments undergoing vasospasm the decrease in resting tension induced by W-7 was markedly diminished (p<0.01), that induced by nicardipine was unchanged, and that induced by H-7 was significantly increased (p<0.01). Our results indicate that spontaneous tonus due to activation of the protein kinase C system is significantly augmented in segments undergoing vasospasm. Thus this system, rather than the Ca 2+ /calmodulin system, appears to play a major role in the occurrence of chronic vasospasm. Received December 20, 1990; accepted May 21, 1991. this contraction to activation of the protein kinase C system because it was inhibited by protein kinase C inhibitors such as H-7 and staurosporine and because it was accompanied by phosphorylation of proteins other than 20-kd myosin light chain. 10 Using the beagle two-hemorrhage SAH model, 11 we have further shown that the basilar artery narrowing due to chronic vasospasm was significantly reversed by the topical application of protein kinase C inhibitors and that there was a highly significant linear correlation between the severity of angiographic narrowing of the basilar artery and the increase in arterial levels of an intrinsic protein kinase C activator throughout the evolution of chronic vasospasm.12 Thus, our preceding results suggest that active smooth muscle contraction due to protein kinase C activation is involved in the occurrence of chronic vasospasm. In this study, we examined whether the in vitro contractile activities of arterial segments excised from the basilar artery during chronic vasospasm would show enhanced activity of the protein kinase C system in response to various agonists. Materials and MethodsWe used 36 beagles, 12, eight, four, and 12 in the first, second, third, and fourth experiments, respecDownloaded from http://ahajournals.org by on April 28, 2019