[Good practices in telemedicine]. / Bonnes pratiques en matière de télémédecine.

Telemedicine, or remote medicine, has become an important tool for health care providers as a result of the SARS-Cov2 pandemic. It must be considered as a tool capable of improving the practice of modern medicine. This text reminds the rules of its practice and encourages the organization of teaching.

Localization of phosphodiesterase-4 isoforms in the medulla and nodose ganglion of the squirrel monkey.

Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.

Pyrogenic reactions associated with single daily dosing of intravenous gentamicin.

OBJECTIVE: To identify risk factors associated with an unexpected outbreak of pyrogenic reactions (PR) following intravenous gentamicin. DESIGN: We conducted two cohort studies. PRs were defined as chills, rigors, or shaking within 3 hours after initiating the gentamicin infusion during the preepidemic (December 1, 1997-January 15, 1998) or epidemic (May 1-June 15, 1998) periods. We tested gentamicin vials for endotoxin using the limulus amebocyte lysate assay. SETTING: Inpatient services of a large community hospital in Los Angeles, California. RESULTS: During the epidemic period, 22 (15%) of 152 patients developed documented PRs following intravenous gentamicin. PRs were more likely among patients receiving single daily dosing (SDD) than multiple daily dosing gentamicin (20/73 [27%] vs. 2/79 [3%]; relative risk, 10.8; 95% confidence interval, 2.6 44.7). Laboratory analysis of gentamicin vials found endotoxin levels that were higher among Fujisawa-brand gentamicin (implicated brand) than gentamicin used after the outbreak terminated (non-implicated brand). Although endotoxin levels in the vials did not exceed US Pharmacopeia limits (1.7 endotoxin units/mg gentamicin), the use of SDD gentamicin may place patients at greater risk of receiving doses of endotoxin above the threshold for PRs in humans. CONCLUSIONS: Reassessment of the acceptable amounts of endotoxin in gentamicin and other parenteral products should be considered when dosing intervals used in clinical practice change.

Retinal and choroidal manifestations of cat-scratch disease.

OBJECTIVE: The ability to diagnose cat-scratch disease (CSD) has been facilitated greatly by the recent isolation and characterization of Bartonella henselae (formerly genus Rochalimaea) and Afipia felis and by the subsequent development of specific enzyme-linked immunosorbent assay (ELISA) serologic tests. This study will help define the patterns of posterior segment ocular involvement in patients with confirmed CSD. DESIGN: The study design is a retrospective case study and literature review. PARTICIPANTS: Two consecutive patients with acute visual loss from retinal manifestations of CSD participated. INTERVENTIONS: The diagnosis was confirmed by B. henselae ELISA testing. Patients underwent extensive medical and ophthalmic investigations to exclude other causes of retinal and choroidal disease. Ophthalmic investigation included fluorescein angiography and visual field testing. One patient received antibiotic therapy with cefotaxime, then with ciprofloxacin, and was treated with oral prednisone. The other patient was improving for several weeks before oral doxycycline was given. MAIN OUTCOME MEASURES: The clinical syndromes observed were studied over time using visual acuity, visual field, and clinical findings. Data were collated with cases from the literature. RESULTS: Unilateral neuroretinitis and an unusual macular retinitis developed in patient 1, as did bilateral small intraretinal white spots and a unilateral choroidal infiltrate that continued to develop while the patient received antibiotic treatment. Patient 2 had a branch arteriolar occlusion in relation to a perivascular retinal infiltrate and a few small, bilateral, intraretinal white spots. There was gradual resolution with visual improvement while the patient received the antibiotic treatment, although therapeutic efficacy could not be determined. Patient 1 also received oral corticosteroids. A detailed analysis of the literature placed these findings in context. CONCLUSIONS: An unusual, well-defined retinal opacification with features of both multiple retinal arteriolar occlusions and a low-grade retinitis was described. Several features also may occur in posterior segment CSD, including neuroretinitis, a retinal white spot syndrome, and focal choroiditis.

Characterization of CHO-K1 cells stably expressing PDE-IV enzymes. Whole-cell cAMP determinations vs broken-cell enzymatic assays.

A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.

Intracellular calcium responses in CF/T43 cells: calcium pools and influx pathways.

We studied the intracellular free Ca2+ concentration ([Ca2+]i) response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine (100 microM; duration approximately 10 min) biphasically increased [Ca2+]i, with a rapid initial peak (30-45 s) followed by a smaller second peak that lasted for several minutes before returning to baseline. Neither peak specifically depended on Ca2+ influx. Exposure to bradykinin (10 microM) elicited a single peak that lasted 3-3.5 min before returning to baseline. Bradykinin increased intracellular inositol 1,4,5-trisphosphate (IP3), which peaked and returned to baseline within 150 s. Histamine also increased IP3 monophasically, but the peak was brief (< 20 s). Both phases of the Ca2+ response to histamine exhibited similar responsiveness to histamine concentration and sensitivity to antagonists. Cimetidine or thioperamide (1 mM) had no effect on the second peak. Pyrilamine blocked the second peak at concentrations similar to those required to block the initial peak. Activation of the second peak was observed at a threshold concentration of 1 microM comparable with the threshold of the initial peak. Neither adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, nor cyclic ADP (cADP)-ribose altered the second phase of the histamine response.

Existence, properties, and functional expression of "Maxi-K"-type, Ca2+-activated K+ channels in short-term cultured hepatocytes.

A large-conductance, Ca2+-activated K+ channel was identified and characterized in embryonic chick hepatocytes using the patch-electrode voltage-clamp technique. The channel conductance was 213 pS in excised patches bathed in symmetrical 145 mM KCI and 1 mM Ca2+. Current-voltage relationships were linear with high K+ on both sides of the membrane but showed constant field rectification as the K+ gradient was increased. The reversal potential shifted 58 mV per 10-fold change in the ratio of external to internal K+. Channel openings occurred at potentials higher than +50 mV in cell-attached patches. The open probability X voltage relationship shifted to more negative potentials in excised, inside-out patches exposed to a solution containing high Ca2+. The voltage sensitivity of the channel was not significantly affected by changes in internal Ca2+ concentration. Conversely, channel gating, reflected in the half-activation potential, shifted 118 mV per 10-fold change in internal Ca2+ at concentrations less than approximately 2 microM, although at higher Ca2+, this parameter was Ca2+ insensitive. Channel open probability in cell-attached patches increased significantly following exposure of the cells to either the Ca2+ ionophore A-23187 or L-alanine, a cell-volume modulator. Channel density increased with time spent in culture from no observations in 10-hr cells, through 13 and 80% of patches in 24-and 48-hr cultured cells, respectively. The implications of delayed functional expression for ion channel studies in acutely dissociated cells is discussed.

One-month exposure to inhaled endotoxin produces a dose-dependent increase in stored mucosubstances in rat intrapulmonary airways.

This study examined the production of stored mucosubtances in rats after repeated exposure to aerosolized endotoxin, a common contaminant of bioaerosols. Male Fischer 344 rats were exposed to aerosolized saline (sham control) or endotoxin (target concentrations of 0.05, 0.5, and 5.0 micrograms/m3) for 3 h/day, 5 days/week for 4 weeks. Following the final exposure, the left lung of each animal was lavaged and the right lung and nasal cavity were fixed with buffered formalin. Morphometric examination of Alcian blue/Periodic acid Schiffs-stained (AB/PAS) lung sections demonstrated dose-dependent increases in stored intraepithelial mucosubstances in the intrapulmonary airways of endotoxin-exposed rats. Threefold and eightfold increases in stored mucosubstances were observed in generation 5 airways of animals exposed to 0.5 or 5.0 microgram/m3 endotoxin, respectively (p < .05). This mucous cell metaplasia in the intrapulmonary airways was not accompanied by evidence of lung inflammation or increased AB/PAS-staining high molecular weight material in lavage fluid. Furthermore, despite significant deposition of endotoxin aerosols (mass median aerodynamic diameter of 1.9 microns) in the nasal cavity, no significant changes in stored mucosubstances were observed in the nasal septum. In animals repeatedly exposed to 5.0 micrograms/m3 endotoxin and allowed to recover for 1 month, stored mucosubstances in the intrapulmonary airway were still more than fivefold greater than control values. Thus, in rats, repeated exposure to inhaled endotoxin produced a persistent mucous cell metaplasia only in the intrapulmonary airways.

Service plans and clinical interventions targeted by the oncology pharmacist.

Pharmacists are faced with a changing health care environment where they are being asked to demonstrate "value-added" to patient care. Because of the toxicity, complexity, intensity, and cost of cancer treatment, oncology pharmacists have opportunities to prevent adverse drug reactions, reduce costs, optimize drug regimens, and improve patient outcomes. Programs implemented by pharmacists practicing on a 40-bed adult inpatient oncology unit are described, including (1) chemotherapy quality improvement, (2) treatment of febrile neutropenia, and (3) management of peripheral blood cell transplant patients.

Effect of dexamethasone on antigen-induced high molecular weight glycoconjugate secretion in allergic guinea pigs.

The ovalbumin-sensitized guinea pig is commonly used as a small animal model of allergic asthma. This animal model exhibits many of the hallmark characteristics observed in patients afflicted with asthma including nonspecific airway hyperreactivity, airway eosinophilia, early and late phase bronchoconstriction, and plasma extravasation into the airways. In addition, mucous hypersecretion in the airways of asthmatic patients is thought to be responsible for the plugging of distal airways and to contribute to the morbidity and mortality associated with the disease process. In this study we examined whether the allergic guinea pig model exhibits an increase in airway high molecular weight glycoconjugate (HMWG) secretion in response to an antigen challenge and whether dexamethasone exerts any modulatory effects upon the response. Ovalbumin (OVA) -sensitized guinea pigs were challenged with OVA 2 wk following the initial exposure. Trachobronchoalveolar lavages (TBAL) were performed, and the samples were assayed for total eosinophil cell number, eosinophil peroxidase activity (EPO), and both acidic and neutral HMWG content. Morphometric analysis of mucous-containing cells was also performed on tissue sections prepared from the trachea, mainstem bronchus, and three lobes of the left lung. Within 24 h of an antigen challenge, TBAL samples obtained from the allergic guinea pigs exhibited increases in eosinophil cell number, measured EPO enzyme activity, and acidic HMWG content compared to TBAL samples prepared from vehicle-exposed animals. These antigen-induced changes were dependent on the concentration of aerosolized OVA administered. Exposing the animals to 0.3% OVA provoked a 6.23-fold increase in airway eosinophils, 15-fold elevation in TBAL EPO enzyme activity, and 175% increase in TBAL acidic HMWG. No significant changes in TBAL neutral HMWG were measured. The changes in measured EPO activity correlated with the levels of acidic HMWG found in the TBAL samples (r = 0.73, P < or = 0.001). The measured increase in TBAL acidic HMWG was time dependent and was found to be maximal at 2 h post-antigen challenge. Morphometric analysis of Alcian blue (pH 2.5) -stained airway sections showed a decline in stored mucosubstances following the antigen exposure, supporting the notion that the allergic guinea pig model exhibits a mucosecretory component. Pretreating the animals with dexamethasone attenuated the antigen-induced release of HMWG and changes in measured EPO activity. In conclusion, these data indicate that the allergic guinea pig may be a useful model for examining the neural and cellular mechanisms underlying mucus hypersecretion in individuals afflicted with bronchial asthma.

Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis.

Chronic exposure of rats to high concentrations of SO2 gas causes pathologic changes in airway similar to those seen in human chronic bronchitis. The purpose of this study was to examine the pulmonary mechanical correlates of these changes and to quantify the extent of mucous hypersecretion by measuring changes in mucous glycoproteins. Female Sprague-Dawley rats were exposed to 250 ppm SO2 gas, 5 h/d, 5 d/wk, for a period of 4 wk. Control rats were exposed to air only. On the day after the last SO2 exposure, rats were anesthetized, instrumented for the measurement of pulmonary resistance (RL) and dynamic compliance (Cdyn), and ventilated. Chronic SO2 exposure caused a small but significant increase in RL and decrease in Cdyn. Airway responsiveness to inhaled aerosolized methacholine was increased in SO2-exposed rats, as indicated by approximately 6.6- and 4.6-fold decreases respectively, in the doses of inhaled methacholine required to double RL or decrease Cdyn to 50% of baseline. SO2 exposure had no effect on the contractile response of the trachea measured in vitro. Tracheae and lungs from SO2-exposed animals exhibited 140 and 535% increases in measured neutral mucous glycoproteins, respectively, and 33 and 37% increases in acid glycoproteins. Our results indicate that this animal model of chronic bronchitis mimics the mucous hypersecretion, airway obstruction, and increased airway responsiveness observed in human bronchitis and may allow us to begin to probe their mechanistic basis.

The inwardly rectifying potassium current of embryonic chick hepatocytes.

The single channel and whole-cell properties of an inward, rectifying potassium current in cultured embryonic chick hepatocytes were studied at 20 degrees C. In cell-attached patches, channels open upon membrane hyperpolarization and are present in about 90% of cell-attached patches. With 145 mM potassium in the pipette, inward current has a slope conductance of 80 pS. The conductance is not a linear function of the external potassium concentration. Current saturates at high external potassium and has a Michaelis-Menten affinity constant of 275 mM potassium. Substitution of gluconate for chloride in the external solution has no significant effect on conductance, and the reversal potential shifts approximately 18 mV with a change in external potassium from 72.5 to 145 mM indicating potassium selectivity. Channel openings are characterized by multiple brief closures during a burst. The channel is inhibited by external cesium in a concentration-dependent manner. Block is characterized by an increased frequency of transient closures. Whole-cell dialysis with 145 mM CsCl of cells bathed in 145 mM KCl reveals time-independent inward currents that reverse at 0 mV in response to 200 msec-voltage steps. Although voltage ramps evoke currents that are 75% potassium dependent and cesium sensitive, the mean chord conductance (425 pS) indicates that less than five channels are open at any instant. We suggest that the inwardly rectifying potassium channel is partially inactivated in the dialysed hepatocyte.

Hyperplastic effects of aerosolized sodium metabisulfite on rat airway mucus-secretory epithelial cells.

The ability of aerosolized sodium metabisulfite to induce hypertrophic and hyperplastic changes in rat airway secretory epithelial cells was investigated. A 10% solution of sodium metabisulfite was aerosolized into a Plexiglas exposure chamber, using an ultrasonic humidifier. The level of SO2 gas generated by this apparatus was measured to be 500 ppm. Measured levels of neutral and acidic mucous glycoproteins in extracts from tracheal and lung tissue were used as indices of hypertrophic (increases in mucus content per cell) and hyperplastic (increased numbers of cells containing mucus per gram of tissue) changes occurring in mucus-secreting cells of the airways. Exposing rats to sodium metabisulfite for 3 weeks resulted in profound increases in total neutral mucous glycoproteins found in tracheal and lung tissue (6.2-fold and 10.1-fold, respectively), compared with the H2O-treated counterparts. Total acidic mucous glycoproteins were significantly elevated in lung tissue only (13.5-fold). In addition, neutral and acidic mucous glycoproteins were elevated 20-fold and 9-fold, respectively, in bronchoalveolar lavage samples prepared from sodium metabisulfite exposed animals. These results indicate that aerosolized sodium metabisulfite may be a useful agent for developing small animal models of mucus hypersecretion.

Leukotriene D4-induced increases in cytosolic calcium in THP-1 cells: dependence on extracellular calcium and inhibition with selective leukotriene D4 receptor antagonists.

Agonist-induced changes in intracellular calcium ion concentration ([Ca++]i) were examined in human monocytic leukemia THP-1 cells loaded with fura 2/acetoxymethyl ester (fura 2/AM). Leukotriene (LT)D4 induced a concentration-dependent biphasic response consisting of a transient phase (up to 5-fold peak increase) followed by a sustained phase, showing characteristics of a receptor-operated calcium channel. Homologous desensitization to LTD4 was observed. The responses to LTD4 were reduced by 80 to 90% in calcium-free buffer. The responses to LTD4 in a calcium-free buffer were dependent upon the duration of prior exposure of the cells to a calcium-free environment. The response at 30 or 60 min after exposure to calcium-free buffer was greater than that at earlier time points (time-dependent sensitization). Similar responses were obtained with THP-1 cells exposed to EDTA-containing buffer. It is speculated that such time-dependent sensitization is a result of changes at the receptor level. The responses to LTD4 were blocked by two specific LTD4 antagonists, MK-0571 and ICI-204,219, in a concentration-dependent manner. When given after addition of LTD4, MK-0571 or ICI-204,219 reversed the sustained phase of the LTD4-induced response, suggesting that maintenance of the response requires persistent activation of the LTD4 receptor. ICI-204,219 was 5 to 10 times more potent than MK-0571 (IC50 values of 1.1 and 9.3 nM, respectively), in agreement with results from radioligand binding studies reported separately.

Hypertrophic and hyperplastic changes of mucus-secreting epithelial cells in rat airways: assessment using a novel, rapid, and simple technique.

Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.

Diphenylamine-2-carboxylate analogues block Cl- conductances in A7r5 cells by affecting cellular Ca2+ homeostasis.

We have investigated the cellular signalling pathway by which vasopressin stimulates a Ca2(+)-dependent Cl- conductance and the effects of two known Cl- channel blockers in cultured rat A7r5 aortic smooth muscle cells using anion efflux and fluorescent Ca2+ imaging studies. Addition of vasopressin (100 nM) to A7r5 cells enhanced 125I (Cl- substitute) efflux from the cells through a V1 receptor-mediated pathway. Maximal increases in the rate of efflux were observed 1 min following addition of vasopressin (4-fold above basal levels). Activation of the V1 pathway was demonstrated by an increase in inositol trisphosphate (IP3) formation and lack of cAMP accumulation by the cells following the addition of vasopressin. Fluorescent ratio imaging with fura-2 revealed that addition of vasopressin to the cells results in an increase of [Ca2+]i which peaks within 20 s and does not return to resting levels during the 100 s observation period. The addition of a Ca2+ ionophore mimicked the vasopressin-induced efflux from the cells. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and a chloro-substituted compound (cpd 149) inhibited the vasopressin-stimulated 125I efflux from the cells. The concentrations of NPPB and cpd 149 required to inhibit 125I efflux from the cells were similar to those which also attenuated vasopressin-induced Ca2+ transients in the cells. NPPB and cpd 149 had no effects on the ionomycin stimulated efflux. The mechanism(s) by which cpd 149 exerts its effect on stimulated efflux was examined by measuring its action on vasopressin-induced changes in IP3. Compound 149 inhibited IP3 generation in response to vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of intrahepatic innervation in caesium-induced haemodynamic oscillations in the rat liver.

1. Cs+ was used to study the mechanisms involved in the induction of haemodynamic and metabolic oscillations in the isolated rat liver perfused by the portal vein. 2. Cs+ (2.25 mM) in K(+)-free perfusate causes the appearance of periodic increases in portal pressure (3.87 +/- 0.6 mmHg, n = 24) and decreases in O2 uptake (111 +/- 26 microM) at a frequency of 0.013 +/- 0.002 Hz. A 19.8 +/- 1.2 min induction time occurs prior to the generation of periodicity. The oscillations are interspersed with low amplitude shoulders and peaks indicating multiple non-phasic components. 3. Cs+ concentrations higher or lower than 2.25 mM decrease oscillation amplitude and render them aperiodic. No oscillations are observed with 0.5 or 12.5 mM Cs+. 4. The oscillations are reversibly blocked by 1 mM K+, 1 mM EGTA, 5 microM verapamil or 0.17 microM tetrodotoxin. One micromolar chlorpheniramine, 10 microM propranolol, 12 microM phentolamine or 35 microM atropine also maximally inhibit the Cs(+)-induced vasoactivity. 5. The results show that the liver perfused by the portal vein has all of the components required to activate vascular synchrony including intrinsic neural input and responsive vasoactive cells.

The Na-K-Cl cotransporter of avian salt gland. Phosphorylation in response to cAMP-dependent and calcium-dependent secretogogues.

The effect of a cAMP-dependent secretogogue (VIP) on the phosphorylation of an endogenous, membrane-bound protein (pp170) was assessed in an intact cell preparation from the avian salt gland. The addition of VIP, in the presence of 100 microM isobutylmethylxanthine, resulted in a concentration-dependent increase in phosphorylation of pp170. This effect was rapid and transient with a 3-5-fold increase in phosphorylation occurring 1 min after the addition of VIP. Under similar incubation conditions, VIP stimulated a 4.6-fold increase in cAMP accumulation that paralleled phosphorylation. Exposure of cells to either forskolin or 8-Br-cAMP resulted in a 5-8-fold increase in the phosphorylation of pp170. The effect of forskolin was dose dependent with an EC50 similar to that for stimulation of secretion (35 nM). These results implicate an involvement for a cAMP-dependent protein kinase in the phosphorylation of pp170. The identity of pp170 was assessed utilizing a monoclonal antibody (Q3) directed against pp170. Q3 recognized a single 170-kDa band on Western blots of salt gland membrane protein. Immunoprecipitation of pp170 from salt gland cells resulted in the selective extraction of a single protein whose phosphorylation state was increased approximately 5-fold in response to carbachol or VIP. The identity of pp170 was established using two criteria. First, Q3 recognized affinity-purified Na:K:Cl cotransporter preparations from shark rectal gland membranes. Second, pp170 was selectively immunoprecipitated by monoclonal antibodies (J3, J4, and J7) that recognize different epitopes of the shark transport protein. These results suggest that pp170 is homologous to the shark rectal gland Na-K-Cl cotransporter, and thus the proteins may be functionally similar.

Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells.

The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.