Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression.

cGMP-based regulatory systems are vital for counteracting the renin-angiotensin system (RAS) which promotes volume expansion and high blood pressure. Natriuretic peptides and nitric oxide acting through their second messenger cGMP normally increase natriuresis and diuresis, and regulate renin release; however, the severe pathological state of cardiac heart failure is characterized by elevated levels of atrial natriuretic peptide that are no longer able to effectively oppose exaggerated RAS effects. There is presently limited information on the intracellular effectors of cGMP actions in the kidney. Recently we reported the cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), which is highly enriched in intestinal mucosa but was also detected for the first time in kidney. In the present study, cGK II was localized to juxtaglomerular (JG) cells, the ascending thin limb (ATL), and to a lesser extent the brush border of proximal tubules. An activator of renin gene expression, the angiotensin II type I receptor inhibitor, losartan, increased cGK II mRNA and protein three to fourfold in JG cells. In other experiments, water deprivation increased cGK II mRNA and protein three to fourfold in the inner medulla where both cGK II, and a kidney specific CI- channel shown by others to be regulated by dehydration, are localized in the ATL. Whereas additional data suggest that cGK I may primarily mediate cGMP-related changes in renal hemodynamics, cGK II may regulate renin release and ATL ion transport.

Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.

Development of germ cell transplants: morphometric and ultrastructural studies.

Mouse-to-mouse transplants were studied at 10 min, 9 h, 24 h, 1 week, 1 month, 2 months, and 3 months post-transplantation. Data from a previous light microscope study were confirmed and extended using morphometric and ultrastructural techniques. As soon as 10 min after introduction of the germ cells from one mouse into the tubule lumen of a recipient mouse they developed relationships with small Sertoli cell processes. The extent of this surface-to-surface relationship increased in animals sacrificed up to 1 week post-transplantation. Most transplanted germ cells retained the characteristics of the donor germ cells after they had been isolated and pelleted. Nearly all transplanted cells eventually underwent phagocytosis by the recipient Sertoli cells. The presence of small apparent clones of germ cells after 1 week of transplantation indicated that some germ cells may divide and survive for short periods within the epithelium. No discernible qualitative subcellular changes in the host Sertoli cell accompanying the development of transplant spermatogenesis were noted. Macrophages were present in the region of the boundary tissue between myoid cells and appeared to increase in number in the peritubular tissue of transplanted testes. Images suggest that they migrated into the tubule to gain entrance to the lumen and there take on the form of activated macrophages. Some macrophages phagocytose sperm at 2 months and 3 months post-transplantation. A testis weight increase previously demonstrate to occur at 24 h post-introduction of germ cells was found to be due to an increase in the volume of the tubular lumen. The increase of lumen size at 24 h was not related to the volume of the injected material. It is suggested that the presence of injected cells, likely germ cells, in the tubule lumen stimulated increased secretion by the Sertoli cell.

Induced ovulation and conception in locating sows.

Fifty lactating sows were injected with 1,500 IU pregnant mare serum gonadotrophin (PMSG) at an average of 25 days postpartum. Twenty-four of these sows received prostaglandin F2 alpha (PGF2 alpha) 24 hr prior to PMSG. Ninety-six hours after the PMSG injection, 1,000 IU of human chorionic gonadotrophin (HCG) were injected. Artificial insemination was performed at 24 and 36 to 42 hr post-HCG. The PMSG/HCG treatment resulted in pregnancy in 17 of 20 sows slaughtered from 34 to 43 days postbreeding and in 23 of 30 sows allowed to complete gestation. Mean numbers of corpora lutea (33) and viable embryos (15) were counted at slaughter. Litter sizes were averaged (11) for those sows allowed to farrow. Treatment with PGF2 alpha prior to PMSG injection had no effect on conception rates, number of corpora lutea, number of embryos or litter size in the lactating sows. In a second experiment, the same hormone treatments were administered to lactating sows beginning on day 5, 10, 15 or 20 postpartum. Pregnancy rates were 0/10, 2/10, 8/10 and 6/10, respectively (P less than .05, chi-square). At slaughter (30 to 40 days postbreeding), corpora lutea and embryo numbers recorded from pregnant sows were 23.0, 9.5; 31.5, 15.3, and 28.0, 18.8, respectively, for the sows in the day 10, day 15 and day 20 groups. In a third experiment, sows were given PMSG-HCG as previously described on either day 5 (five sows) or day 10 (14 sows) postpartum. Laparotomy of these sows 2 to 5 days postbreeding revealed minimal ovarian responsiveness at day 5, but 43% of the animals responded with multiple ovulations at day 10. The low pregnancy rate seen at day 10 in Exp. 2 may reflect embryonic mortality due to unfavorable uterine environment. We conclude that the PMSG/HCG treatment followed by timed artificial insemination of lactating sows will induce ovulation and coneption as early as 15 days postfarrowing. Pregnancy is thus concurrent with lactation, eliminating the need for early weaning and reducing the interval between successive farrowings.

Experimental infection of guinea pigs with varicella-zoster virus.

Weanling guinea pigs are susceptible to infection with varicella-zoster virus (VZV). Animals inoculated intranasally or subcutaneously with VZV grown in fetal guinea pig tissue culture shed virus from the nasopharynx and seroconverted to VZV. Viremia occurred in some animals. Animal-to-animal transmission of VZV was observed. Infection of weanling guinea pigs with VZV should allow assessment of the pathophysiology of viral infection in immunocompetent and specifically immunologically modified animals.

Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.

Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.

Differential expression of the cytotoxic and hemolytic activities of the ApxIIA toxin from Actinobacillus pleuropneumoniae.

The ApxIIA protein secreted from Actinobacillus pleuropneumoniae is both hemolytic and cytotoxic. However, when the cloned apxII operon is expressed in Escherichia coli, two forms of the ApxIIA protein can be recovered. Toxin which remains intracellular has hemolytic and cytotoxic activities, while toxin that is secreted is cytotoxic with little or no hemolytic activity. This indicates that the cytotoxicity of ApxIIA is independent of its hemolytic activity.

Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II.

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.