Mechanisms of alcohol impairment of recovery from mechanically denuded areas made within cultured rat hepatocytes.

The effect of ethanol on the ability of a denuded hepatocyte cell surface to recover is unknown. We therefore determined the effect of ethanol on the rate of renewal of mechanical wounds made in near-confluent monolayers of primary cultures of rat hepatocytes. We found that ethanol exerted a dose-dependent effect to impair rat hepatocyte recovery and that at least 12 hours of exposure to ethanol was required to induce this impairment. The effect of ethanol to impair recovery of denuded areas of epithelium was not seen in two established renal tubular epithelial cell lines. Ethanol impairment of rat hepatocyte recovery could be blocked by an alcohol dehydrogenase inhibitor (4-methyl pyrazole) and potentiated by an inhibitor of aldehyde dehydrogenase (pargyline). The effect of ethanol to inhibit rat hepatocyte recovery of denuded areas was not associated with any change in hepatocyte cell surface expression of the beta1 integrin subunit as determined by flow cytometry. These results suggest that acetaldehyde is responsible for ethanol inhibition of hepatocyte recovery from mechanical injury and that this impairment occurs independent of cell surface expression of the beta1 integrin subunit.

Mechanisms of recovery from mechanical injury of cultured rat hepatocytes.

The mechanism(s) whereby hepatocytes restore denuded areas remains unknown. We therefore studied the recovery of denuded areas made in monolayers of primary cultures of rat hepatocytes. Minimal recovery occurred in cells plated on plastic. Plating on Matrigel produced modest recovery (25% at 24 h), whereas plating on a type I collagen substrate resulted in > 70% recovery at 24 h. The rate of recovery on collagen could be attenuated by a monoclonal antibody directed against the extracellular domain of the beta 1-integrin subunit. Monoclonal antibodies directed against CD44 (the hyaluron receptor) and E-cadherin did not influence the rate of recovery. Recovery could be stimulated, in a dose-dependent fashion, by epidermal and hepatocyte growth factors. The effects of epidermal and hepatocyte growth factors to promote recovery occurred in the absence of 5-bromo-2'-deoxyuridine uptake, suggesting a proliferation-independent mechanism. Transforming growth factor-beta 1 inhibited recovery. Exposure to selected cytokines (interleukins 1 and 2), an adenine nucleotide [adenosine 5'-O-(3-thiotriphosphate)], adenosine, pertussis toxin, and selected agents that bind to fibronectin and other matrix component adhesive sites (heparin and the RGD peptide) did not influence the rate of recovery of hepatocytes. However, the peptide DGEA, which can bind to collagen adhesive sites, attenuated recovery. These studies demonstrate that primary cultures of rat hepatocytes require a particular type of extracellular matrix to renew denuded areas and that the beta 1-integrin subunit may be involved in this recovery process. Hepatocyte recovery of denuded areas can be modulated by growth factors in both a stimulatory (epidermal and hepatocyte growth factors) and an inhibitory (transforming growth factor-beta 1) fashion.

Adenine nucleotide and protein kinase C regulation of renal tubular epithelial cell wound healing.

The present studies were done to determine the effect of selected adenine nucleotides on healing of wounds made by mechanically denuding areas in confluent monolayers of renal tubular epithelial cells. We found that hydrolyzable and nonhydrolyzable forms of ATP but not UTP stimulated healing of LLC-PK1 cell wounds, while both ATP and UTP promoted healing of MDCK cell wounds, suggesting that different subtypes of purinoceptors regulated wound healing in these cells. Stimulation of wound healing by ATP was equivalent in control cells and in cells in which irradiation suppressed proliferation, suggesting a prominent role for cell migration in the healing process. Since ATP receptors are often linked to activation of protein kinase C, the effect of a protein kinase C activator (4 beta-phorbol 12-myristate 13-acetate, PMA) on wound healing was studied. Long-term (24 hr) exposure to PMA inhibited while short-term (30-120 min) exposure to PMA enhanced cell wound healing. Two chemically and mechanistically dissimilar protein kinase C inhibitors (calphostin C and chelerythrine) inhibited LLC-PK1 and MDCK cell wound healing, and calphostin C prevented ATP enhancement of LLC-PK1 healing. These observations suggest a role for protein kinase C in regulation of basal and adenine nucleotide-stimulated wound healing. Adenosine triphosphate did not affect cell-cell adhesion of either LLC-PK1 or MDCK cells. Adenine nucleotides and PMA enhanced and calphostin C inhibited short-term adhesion of LLC-PK1 and MDCK cells to plastic and to other substrates such as fibronectin, laminin and collagen type IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of recovery from mechanical injury of renal tubular epithelial cells.

The mechanism(s) whereby a denuded renal tubular epithelial cell surface becomes reestablished remains unknown. We therefore measured the rate of renewal of mechanical wounds made in confluent monolayers of two established renal tubular epithelial cell lines. We found that wounds of MDCK cells heal at a faster rate than wounds of LLC-PK1 cells. The magnitude of wound healing did not differ when cells grown on plastic were compared with cells grown on fibronectin, laminin, or collagen. Irradiation (4,000 rads) of MDCK and LLC-PK1 cells significantly reduced indexes of proliferation (5-bromo-2'-deoxyuridine and thymidine uptake) without affecting wound healing. Serum and epidermal growth factor (EGF) enhance whereas transforming growth factor-beta 1 (TGF-beta 1) impairs wound healing. Hepatocyte growth factor (HGF) stimulates wound healing at low concentrations and inhibits healing at high concentrations in MDCK cells while not affecting healing of LLC-PK1 cell wounds at any concentration. Several interleukins (IL-1, IL-2, IL-3, and IL-6) did not affect wound healing in either cell type. Healing of LLC-PK1 but not MDCK cells was impaired by exposure to a peptide containing a RGD sequence. Conversely, healing of MDCK but not LLC-PK1 cells was impaired by the REDV tetrapeptide. Healing of both LLC-PK1 and MDCK was impaired by heparin but not by the LDVPS peptide. These results demonstrate that mechanical wounds of LLC-PK1 and MDCK cells heal, at least in part, by migration. Healing is regulated by serum and growth factors including EGF, HGF, and TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Escape from the antiproliferative effect of transforming growth factor-beta 1 in LLC-PK1 renal epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) usually inhibits proliferation of epithelial cells. We find that LLC-PK1 renal tubular epithelial cells develop rapid in vitro resistance to the inhibitory effects of TGF-beta 1 and subsequently proliferate in response to TGF-beta 1. This unique response to TGF-beta 1 is not observed in another renal tubular epithelial cell line (MDCK cells). The proliferative response to TGF-beta 1 is additive to that produced by other growth factors. The proliferative response to TGF-beta 1 occurs despite an effect of TGF-beta 1 to suppress epidermal growth factor stimulated c-myc mRNA as determined by Northern analyses. These results suggest that LLC-PK1 cells develop rapid resistance to TGF-beta 1 inhibition of proliferation in vitro and that this resistance occurs despite continued suppression of c-myc mRNA.

Transforming growth factor-beta 1 regulation of signal transduction in two renal epithelial cell lines.

The present studies examine the effect of transforming growth factor-beta 1 (TGF-beta 1) on signal transduction pathways in two cultured renal epithelial cell lines. TGF-beta 1 promotes basal and agonist-stimulated adenylate cyclase activity in LLC-PK1 but not MDCK cell membranes. TGF-beta 1 stimulation of LLC-PK1 membrane adenylate cyclase activity occurs quickly and can be attenuated by pertussis toxin pretreatment. Both TGF-beta 1 and adenosine 3',5'-cyclic monophosphate (cAMP) exert comparable effects on [3H]thymidine uptake in LLC-PK1 cells, suggesting that TGF-beta 1 regulation of adenylate cyclase activity potentially plays a role in mediating biological responses to TGF-beta 1. The activities of protein kinase C and phospholipase A are not affected by TGF-beta 1 in either LLC-PK1 or MDCK cells. Both TGF-beta 1 and epidermal growth factor (EGF) increase expression and induce the appearance of new forms of the cAMP response element binding protein (CREB) in LLC-PK1 cells. These effects of TGF-beta 1 and EGF on CREB appear to be specific since neither TGF-beta 1 nor EGF alters expression of an activating transcription factor in LLC-PK1 cells. The effect of TGF-beta 1 and EGF to alter expression of CREB does not affect CREB binding to its regulatory element in LLC-PK1 cell lysates. These results suggest that some of the biological effects of TGF-beta 1 may be attributed to stimulation of adenylate cyclase activity and cAMP formation as well as to enhanced expression and/or modification of the CREB transcription factor in LLC-PK1 cells.

Cytokine regulation of adenylate cyclase activity in LLC-PK1 cells.

Although several cytokines have been demonstrated to exert pleiotropic responses, there is little information on cytokine regulation of renal tubular epithelial cell function. In the present studies, we find that both T cell-derived (tumor necrosis factor-beta and interleukins 2 and 3) and monocyte/macrophage derived (tumor necrosis factor alpha and interleukin 1 beta) cytokines promote basal, arginine vasopressin- and forskolin-stimulated adenylate cyclase activity in cultured LLC-PK1 cells. No effect of TNF, IL-1 beta, and IL-2 to stimulate protein kinase C activity was observed. TNF-beta, IL-1 beta and IL-2 also modestly stimulated 3H release from 3H-arachidonic acid labeled cells. Mepacrine, a phospholipase A inhibitor, prevented TNF-beta stimulation of 3H release from 3H-arachidonic acid labeled cells and TNF-beta potentiation of adenylate cyclase activity. TNF-beta potentiation of adenylate cyclase activity and stimulation of 3H release from 3H arachidonic acid labeled cells was not prevented by pertussis toxin. These results demonstrate that several cytokines can stimulate adenylate cyclase activity while not affecting protein kinase C activity in cultured renal tubular epithelial cells. The effect of TNF-beta to stimulate adenylate cyclase appears to occur independent of pertussis toxin-sensitive substrate and may involve activation of phospholipase A.

cAMP stimulates protein kinase C activity in cultured renal LLC-PK1 cells.

Exposure of intact LLC-PK1 cells to the phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) increases basal, arginine vasopressin-stimulated, and forskolin-stimulated adenylate cyclase activity in LLC-PK1 membranes. This observation suggests that protein kinase C can increase adenosine 3',5'-cyclic monophosphate (cAMP) in LLC-PK1 cells. To determine whether cAMP regulates protein kinase C activity in LLC-PK1 cells, intact cells were exposed to either forskolin or to soluble cAMP analogues. Acute (5 and 30 min) exposure to either forskolin or cAMP analogues increases protein kinase C activity as observed by two different methods for measuring protein kinase C. Acute exposure to PMA translocates protein kinase C from a soluble to a particulate cell fraction, whereas acute exposure to cAMP increases both soluble and particulate forms of protein kinase C. Longer exposure (18 h) to PMA results in a loss of protein kinase C activity, whereas 18-h exposure to cAMP results in a further increase in protein kinase C activity. The effect of cAMP but not of PMA to stimulate protein kinase C activity can be attenuated by the pro-R diastereoisomer of adenosine 3',5'-cyclic phosphorothioate, suggesting a protein kinase A-mediated effect. These results suggest the presence of a monodirectional mode of signal transduction system interaction in LLC-PK1 cells in which protein kinase C and protein kinase A can potentiate each other.

Regulation by forskolin of cyclic AMP phosphodiesterase and protein kinase C activity in LLC-PK1 cells.

Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.

ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.

In cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.

Effects of kinins on cultured arterial smooth muscle.

The present study uses various kinin agonists and antagonists to examine the cellular mechanisms of bradykinin's actions on intracellular calcium, prostaglandins, and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cultured arterial smooth muscle cells (casmc) obtained from rat mesenteric arteries. Exposure to bradykinin produced a rapid release of calcium (peak less than or equal to 20 s) from intracellular stores and an increase in prostaglandin (PG) E2 and cAMP production in casmc. Compared with bradykinin, the bradykinin B1-agonist [des-Arg9]BK produced only a small increase in intracellular calcium. The bradykinin-mediated increase in intracellular calcium was competitively blocked by the B2 receptor antagonist [D-Arg-O-Hyp3-Thi5,8-D-Phe7]BK (B4307) but not the B1-antagonist ([des-Arg9-Leu8]BK). In addition, the similarity of the dose-response curves for the bradykinin-mediated increase in Ca2+, PGE2, and cAMP (half-maximal stimulation of 12, 11, and 13 nM, respectively) and the ability of the B2-antagonist (B4307) to block each of these effects of bradykinin suggest that all three effects are mediated by the same bradykinin (B2) receptor. Further studies revealed that increases in intracellular calcium are necessary for the bradykinin-mediated increase in PGE2 formation and the subsequent PGE2-dependent formation of cAMP. Taken together, these results suggest that bradykinin acts via a B2-receptor on arterial smooth muscle cells to release calcium from intracellular stores, leading to increases in PGE2 production and the PGE2-dependent activation of adenylate cyclase.

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.

Bradykinin activates protein kinase C in cultured cortical collecting tubular cells.

Bradykinin inhibits vasopressin-stimulated water transport in cortical collecting tubular cells. The biochemical mechanism of this effect was explored by means of primary cultures of rabbit cortical collecting tubular cells. Bradykinin was found to produce a rapid release of calcium from intracellular stores, an increase in sn-1,2-diacylglycerol levels, and a fivefold increase in membrane-bound protein kinase C activity, consistent with stimulation of phospholipase C and activation of protein kinase C in rabbit cortical collecting tubular cells. In addition, bradykinin produced a dose-dependent 46% inhibition of vasopressin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Pretreatment with the protein kinase C inhibitors, H-7 and staurosporine, reversed the bradykinin-mediated inhibition of vasopressin-stimulated cAMP accumulation. In contrast, pretreatment with either the phospholipase A2 inhibitor, mepacrine, or pertussis toxin did not prevent the inhibitory effect of bradykinin on vasopressin-stimulated cAMP production, suggesting that the effects are not mediated by prostaglandin E2 or activation of a pertussis-toxin sensitive guanine nucleotide regulatory protein (e.g., Gi). Because bradykinin also inhibits isoproterenol-stimulated cAMP formation but does not inhibit either basal-, forskolin-, or cholera toxin-stimulated cAMP accumulation, the site of this inhibition appears to involve the hormone receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory subunit (Gs). The results demonstrate that bradykinin stimulates phospholipase C leading to activation of protein kinase C, which then inhibits vasopressin-stimulated cAMP production at the level of the hormone receptor or coupling of the receptor to Gs in cultured cortical collecting tubular cells.

Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells.

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia.

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane-like material adjacent to the dish. Differentiated properties characteristic of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK-ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT-specific anti-keratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular epithelia in the kidney.

A new method for studying human polycystic kidney disease epithelia in culture.

Human polycystic kidney disease (PKD) epithelia were successfully grown in culture and expressed abnormal characteristics. Cysts lining epithelia of superficial and deep cysts were microdissected and compared to individual normal human proximal straight tubules (PST) and cortical collecting tubules (CCT) grown in defined media. PKD cyst epithelia differed from normal renal tubular epithelia in growth patterns and structural and functional properties. PKD epithelia grew more rapidly and showed cyst-like areas in otherwise confluent monolayers. Polygonal and elongate cells contained an epithelial-specific cytokeratin antigen and had polarized morphology. An extremely abnormal basement membrane morphology was seen and consisted of some banded collagen and numerous unique blebs or spheroids. These blebs were apparently extruded from intracellular vacuoles and stained with ruthenium red, suggesting a proteoglycan component. Cytochemistry of marker enzymes demonstrated the presence of NaK-ATPase and alkaline phosphatase, but a lack of gamma-glutamyl transpeptidase. The response of adenylate cyclase activity to vasopressin, parathyroid hormone, and forskolin was significantly diminished in PKD cells as compared to PST and CCT. These studies suggest a defect in cell growth and basement membrane synthesis in human PKD. Cultured PKD epithelia provide a new tool for the study of the pathogenesis of this disease.

Defined human renal tubular epithelia in culture: growth, characterization, and hormonal response.

Individually microdissected nephron segments of defined epithelial origin from human kidneys were cultured in vitro in the present studies. Nephron segments of proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb of Henle, and cortical collecting tubule were grown in defined media. Each cell type retained differentiated characteristics as assessed by ultrastructural morphology, marker enzyme profiles, and adenylate cyclase response to selected hormones. These studies demonstrate the feasibility of using primary cultures of well-defined segments of the human nephron to study human renal tubular epithelia in vitro.

Bluetongue virus in bovine semen: viral isolation.

Vero cell cultures and embryonating chicken eggs were used for direct isolation of bluetongue virus from cattle blood and from semen samples. Cell culture and embryonating chicken eggs each were more effective than was the blood autograft inoculation of susceptible sheep with selected blood and semen samples. Evaluation of the cell culture technique indicated that the quality of the distilled water was the primary factor responsible for the increased sensitivity of the Vero cell cultures for the present blue-tongue viral isolations. Test results showed that urine was a poor specimen for viral isolation when assayed in chicken eggs. A comparison of tests for precipitating and complement-fixing antibodies to bluetongue virus indicated that the precipitin test was the more accurate of the two tests.

Transmission of two strains of epizootic hemorrhagic disease virus in deer by Culicoides variipennis.

Two strains of epizootic hemorrhagic disease virus (EHDV), New Jersey (NJ) and Kentucky (KY), of deer were biologically transmitted between white-tailed deer, Odocoileus virginianus, by Culicoides variipennis. The KY strain, isolated from C. variipennis collected during an epizootic in deer, was identified as EHDV by serological tests. Deer exposed to the KY or the NJ strains of EHDV developed an acute hemorrhagic disease; most deer died 6 to 13 days after infection. Sheep inoculated with EHDV developed no clinical signs of disease.