Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluids and Secretions”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMed1976

Excretion of alkaloids by malpighian tubules of insects.

Nicotine is transported at high rates by Malpighian tubules of larvae of Manduca sexta, Pieris brassicae and Rhodnius prolixus and the transport persists in the absence of alkaloid from the diet. In the fluid-secreting portion of Rhodnius tubules this transport is not coupled to ion transport, nor is it dependent on the physiological state of the animal. The transport, which can occur against a steep electrochemical gradient, shows saturation kinetics with a maximal rate of 700 pmol. min-1 per tubule and is half saturated at 2-3 mM. Nicotine transport independent of ion movements also occurs in the lower resorptive parts of Rhodnius tubules. Both portions of Rhodnius tubules can transport morphine and atropine. These alkaloids and nicotine compete with one naother and are presumed to be carried by the smae transport system. Nicotine transport in Rhodnius was unaffected by organic anions, such as amaranth and benzyl penicillin, or by the organic anion transport inhibitor, probenecid. Fluid secretion in 5-HT-stimulated tubules was reduced by atropine and nicotine, probably by blocking the 5-HT receptors. The Malpighian tubules of adult Calliphora erythrocephala and Musca domestica remove nicotine from bathing solutions, an unknown metabolic accumulating in the tubules. Adult P. brassicae and M. sexta do not exhibit transport of nicotine by their Malpighian tubules.

Alkaloids↗
PubMed2001

Improvement of corneal barrier function by the P2Y(2) agonist INS365 in a rat dry eye model.

PURPOSE: Because purinoceptor P2Y(2) receptor agonists elicit increases in net Cl, fluid transport, and glycoprotein release onto the ocular surface, they are candidates for treatment of dry eye syndrome. Accordingly, the effects of such an agonist INS365 on these parameters were characterized in a rat dry eye model. METHODS: An SD rat dry eye model was used in which exorbital lacrimal gland extirpation decreased the Schirmer test score by at least 50%. After 8 weeks, when significant increases occurred in corneal epithelial permeability, INS365-containing eye drops were applied six times daily for the next 4 weeks at concentrations from 0.03% to 3.0%. Corneal barrier function was evaluated based on measurements with a modified anterior fluorometer of fluorescein penetrance at 1, 2, and 4 weeks after initial application. After INS365 application, the periodic acid-Schiff reagent (PAS)-stained area was evaluated in histologic sections of the tarsal and bulbar conjunctiva. RESULTS: Ten minutes after INS365 eye drop application at doses of either 3. 0% or 8.5%, a 1.5-fold transient increase in tear fluid secretion occurred in both the control and dry eye model animals. These transient increases nearly returned to baseline after 60 minutes. Furthermore, after 5 minutes, 1.0% INS365 was sufficient to cause a maximal transient decrease in the PAS-stained area of more than 30%, which thereafter recovered toward the initial level. Beginning at 2 weeks and continuing for an additional 2 weeks, maximal declines in dye penetrance of approximately 50% occurred with doses of INS365 as low as 1%. Such improvement in corneal epithelial resistance was accompanied by complete restoration of the PAS-stained area to the level seen in the control animal. CONCLUSIONS: In a rat dry eye model, the P2Y(2) agonist INS365 was found to improve surface health, based on increases in tear fluid secretion, corneal epithelial resistance, and release of glycoprotein-containing moieties from goblet cells. These effects suggest that INS365 is a potential therapeutic agent for use in the treatment of dry eye syndrome.

Animals↗
PubMed2002

Loss of hyperpolarization-activated Cl(-) current in salivary acinar cells from Clcn2 knockout mice.

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl(-) current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl(-) current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2(-/-) mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2(-/-) mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2(-/-) mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl(-) channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.

Animals↗
PubMed1995

Carbachol, substance P, and phorbol ester promote the tyrosine phosphorylation of protein kinase C delta in salivary gland epithelial cells.

The initiation of saliva formation by parotid acinar cells, which comprise the majority of cells in this salivary gland, is initiated by the release of neurotransmitters (acetylcholine, substance P) from parasympathetic nerves. In response to substance P and the muscarinic agonist carbachol, two ligands that activate phospholipase C-linked receptors, which stimulate fluid secretion, PKC delta was phosphorylated on tyrosine residues. The maximal agonist-dependent tyrosine phosphorylation occurred within seconds of the addition of either agonist and then returned rapidly to a smaller increased level. Phorbol ester also caused a rapid increase in tyrosine phosphorylation, which reached a maximal level 5 min after the addition of phorbol 12-myristate 13-acetate. The increase in tyrosine phosphorylation of PKC delta was blocked by tyrosine kinase inhibitors genistein and staurosporine. Ionophore-mediated elevation of [Ca2+]i or activation of the beta-adrenergic receptor, epidermal growth factor receptor, or insulin receptor did not promote the tyrosine phosphorylation of PKC delta. These results indicate that tyrosine phosphorylation plays a role in early signal transduction events promoted by the activation of muscarinic and substance P receptors and suggests that the tyrosine phosphorylation of PKC delta has a role in the activation of fluid secretion by neurotransmitters binding to phospholipase C-linked receptors.

Alkaloids↗
PubMed1987

Transcellular sodium fluxes and pump activity in Necturus gall-bladder epithelial cells.

1. Transepithelial Na transport in Necturus was determined by measuring the rate of isotonic fluid flow. The rate at 20 degrees C was equivalent to 175 pmol cm-2 s-1. 2. Ouabain was effective in Necturus, binding to the Na pump in gall-bladder cells with a mean rate constant of 5.4 X 10(3) M-1 s-1. Measurement of the diffusive time constant of the free space for [3H]ouabain shows that the pump must be fully inhibited within 20 s when ouabain is applied to the serosa at 10(-3) M. 3. The serosal Na efflux from loaded cells was inhibited 36% by ouabain equal to a flux of 73 pmol cm-2 s-1. The remaining flux could not be attributed to either exchange diffusion or electrodiffusion induced by ouabain. 4. The transepithelial potential was 0.3 mV serosa positive. The short-circuit current measured was 6.33 +/- 1.9 microA cm-2, equal to a positive univalent ion flux of 65.6 pmol cm-2 s-1 or 38% of the net Na transfer. The current was inhibited within 1-5 min by 5 X 10(-5) M-amiloride. 5. Fluid secretion was immediately inhibited 34% by ouabain, equivalent to an isotonic transport of Na of 59.7 pmol cm-2 s-1. Thereafter it continued for at least an hour, sometimes declining slowly. Amiloride had little effect (13%). 6. The Na pump rate was measured by titrating the cell content with tracer Na at different times after ouabain treatment. The initial slope was equal to a rate of 61.6 pmol cm-2 s-1 or 35% of the net flux at time zero. 7. The Na pump rate has also been measured by recording the rise in cell Na activity with ion-specific micro-electrodes, and correcting for swelling effects. The Na pump rate was very similar to that estimated from the rise in tracer Na content, equal to 59.3 pmol cm-2 s-1 or 31.4% of the transepithelial rate. Examination of the same experiment in the literature shows a closely similar value, about one-third of that expected from fluid secretion or net flux measurements. 8. A scheme is proposed to explain the results, which requires a flow of NaCl through a parallel pathway of small Na content involving exchange en route with the cytoplasmic Na.

Action Potentials↗
PubMed1996

Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line.

The modulation of ion fluxes across the plasma membrane of epithelial cells is central for fluid secretion and absorption. Their disruption can lead to pathological states. An example is cystic fibrosis (CF), a disease characterized by abnormal functioning of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-modulated chloride channel. Here we report the characterization of calcium-activated, DIDS sensitive chloride current and non-selective calcium-activated cation channels in a novel human pancreatic duct cell line (YHV-1) derived from a non-delta F508 mutation CF patient bearing a severe phenotype. Southern blot analysis of the CFTR gene indicates a distinct electrophoretic pattern for the region spanned by exons 15-24, a result presumably related to a mutation which has yet to be identified. In contrast to large calcium-activated chloride currents there were no cAMP-dependent CFTR-type chloride currents. Non-selective cation channels were blocked by intracellular ATP and activated by intracellular calcium and cAMP. We propose the cell line YHV-1 as a suitable model for studying pancreatic ion and fluid secretion alterations in CF.

Blotting, Southern↗
PubMed1996

cAMP promotes gap junctional coupling in T84 cells.

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Cell Line↗
PubMed1985

Dibutyryl-cAMP increases basolateral sodium conductance of mosquito Malpighian tubules.

Dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) stimulates fluid secretion in isolated Malpighian tubules of the mosquito Aedes aegypti. In the present study the effects of cAMP on the basolateral membrane were studied with conventional microelectrodes. Membrane conductances were evaluated from the changes of the basolateral membrane potential (Vbl) consequent to ion changes in the bath. Under control conditions, Vbl measured -65.2 +/- 1.5 mV [83 impalements, 67 tubules]. A fivefold decrease in the bath Na concentration hyperpolarized Vbl by 10.2 +/- 0.6 mV [7], whereas a 4.4-fold increase in the bath K concentration depolarized Vbl by 7.9 +/- 1.0 mV [9]. In the presence of cAMP (10(-4) M) Vbl depolarized to -24.8 +/- 2.7 mV [9]. Vbl now hyperpolarized by 22.7 +/- 1.5 mV [7] for the bath Na change and depolarized by only 3.8 +/- 1.1 mV [6] for the bath K change. Thus the dominant effect of cAMP is the increase of the basolateral membrane Na conductance. This increase is consistent with 1) the depolarization of Vbl and 2) the hyperpolarization of the transepithelial voltage, the decrease of the transepithelial resistance, and the increase of Na and fluid secretion observed previously. Spontaneous oscillations of Vbl were observed and could not be attributed to cyclical changes of the basolateral membrane Na conductance.

Aedes↗
PubMed1993

Membrane potential regulates Ca2+ uptake and inositol phosphate generation in rat sublingual mucous acini.

In salivary acinar cells, muscarinic-induced fluid secretion is associated with a 1,4,5-IP3 induced increase in the cytosolic free Ca2+ concentration ([Ca2+]i), which in turn activates Ca(2+)-dependent K+ and Cl- channels that modulate the membrane potential. In the present study the influence of the membrane potential on [Ca2+]i and inositol phosphates was monitored in rat sublingual mucous acini. Depolarization induced by switching from 5.8 mM extracellular K+ ([K+]e) to 116 mM [K+]e resulted in a transient increase in the [Ca2+]i measured using the Ca2+ sensitive fluorescent indicator Fura-2. This initial rapid (t1/2 approximately 5 s) increase (approximately 3-fold) in [Ca2+]i was dependent on extracellular Ca2+, insensitive to nifedipine, and followed by establishment of a 'new' resting [Ca2+]i, approximately 35% higher than the level in physiological [K+]e. Depolarization also induced a significant rise in the resting cellular inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) contents, but not 1,4,5-IP3 content. Stimulation with 10 microM carbachol (CCh, a muscarinic agonist) produced a biphasic increase in [Ca2+]i, the initial transient phase due to mobilization of Ca2+ from an intracellular pool, and a sustained phase mediated by an influx of Ca2+. Membrane depolarization had no effect on the initial phase, while, the sustained increase in [Ca2+]i was eliminated. The CCh-enhanced quench of the Fura-2 signal by Mn2+ (an index for divalent cation entry) was reversibly inhibited by depolarization. The enhanced Mn2+ uptake induced by inhibiting microsomal Ca(2+)-ATPase with thapsigargin was similarly inhibited by membrane depolarization, consistent with the effect of depolarization primarily acting on the Ca2+ entry pathway and not on receptor coupling. Depolarization did not alter the initial CCh-induced increases in IP3, IP4 or 1,4,5-IP3 content, or the sustained increase in 1,4,5-IP3, whereas, depolarization significantly blunted (> 70%) the sustained, CCh-induced generation of IP3 and IP4. The membrane potential, therefore, appears to modulate Ca2+ activated fluid secretion by controlling the driving force for Ca2+ entry via a depletion-activated Ca2+ entry pathway. Inositol phosphate metabolism is also influenced by the membrane potential, but this effect apparently plays a minor role in regulating [Ca2+]i since 1,4,5-IP3 levels were unchanged by depolarization.

Animals↗
PubMed2001

Acetylcholine-evoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjögren's syndrome.

Recent evidence has indicated that the salivary gland dysfunction associated with Sjögren's syndrome (SjS) is not necessarily due to immune-mediated destruction of acinar tissue. SjS sufferers may possess substantial reserves of acinar tissue but nevertheless be incapable of maintaining salivary flow rates in the normal range. We have investigated the ability of isolated labial gland acinar cells from SjS patients to fluid secrete by measuring agonist-evoked changes in intracellular Ca(2+) ([Ca(2+)](i)) using fura-2 microfluorimetry and activation of K(+) and Cl(-) channels using the patch-clamp whole cell technique. We can confirm that stimulation with a super-maximal dose of acetylcholine (ACh) increased [Ca(2+)]i equally in both control acinar cells and those derived from SjS patients. However, at submaximal concentrations, the dose-response curve for ACh was shifted to the right by approximately one order of magnitude in acinar cells from SjS patients compared to control acinar cells. Patch-clamp measurements consistent with the presence of Ca(2+)-activated K(+) and Cl(-) conductances were obtained from both control acinar cells and those obtained from SjS patients. Dose-dependent activation of the ion channels by acetylcholine was also right-shifted in acinar cells from SjS patients compared to control cells. Our data show that labial gland acinar cells from SjS patients were capable of responding to agonist stimulation by mobilizing [Ca(2+)](i) and activating K(+) and Cl(-) channels consistent with the requirements of fluid secretion. However, the persistent loss of sensitivity to ACh observed in from SjS patients may account for the lack of saliva production observed in these patients in vivo.

Acetylcholine↗
PubMed1998

Involvement of protein kinases and phosphatases in tyrosine phosphorylation of PKCdelta in rat parotid acinar cells exposed to secretory stimuli.

The aim of these studies was to examine the involvement of tyrosine phosphorylation in the signal transduction pathways and secretory events that are promoted by receptor agonists acting on rat parotid acinar cells. Fluid secretion by parotid acinar cells is initiated by the binding of neurotransmitters to GTP(G)-protein-coupled receptors that are linked to phospholipase C, which hydrolyzes phosphatidlyinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Although growth factors produce large changes in tyrosine phosphorylation of multiple proteins involved in proliferation and other cellular processes, tyrosine phosphorylation is not considered to be a general phenomenon of G-protein-coupled receptor activation. However, our results shown that carbachol (a muscarinic acetylcholine receptor agonist), and ligands to other phospholipase C-linked receptors, promoted a rapid increase in the tyrosine phosphorylation of protein kinase Cdelta (PKCdelta), a member of the PKC family of proteins. Phorbol 12-myristate 13-acetate, which binds to the site on PKCdelta to which the endogenous activator sn-1,2-diacylglycerol binds, also increased the tyrosine phosphorylation of PKCdelta. Genistein and staurosporine, two protein kinase inhibitors, blocked the tyrosine phosphorylation of this protein. Thus, PKCdelta becomes tyrosine phosphorylated in response to receptor activation, and this event appears to involve both diacylglycerol production and protein tyrosine kinase activity. This may contribute to early physiological events, including alterations in fluid secretion, that are initiated by neurotransmitters acting on the parotid salivary gland.

Animals↗
PubMed2001

Local uncaging of caged Ca(2+) reveals distribution of Ca(2+)-activated Cl(-) channels in pancreatic acinar cells.

In exocrine acinar cells, Ca(2+)-activated Cl(-) channels in the apical membrane are essential for fluid secretion, but it is unclear whether such channels are important for Cl(-) uptake at the base. Whole-cell current recording, combined with local uncaging of caged Ca(2+), was used to reveal the Cl(-) channel distribution in mouse pancreatic acinar cells, where approximately 90% of the current activated by Ca(2+) in response to acetylcholine was carried by Cl(-). When caged Ca(2+) in the cytosol was uncaged locally in the apical pole, the Cl(-) current was activated, whereas local Ca(2+) uncaging in the basal or lateral areas of the cell had no effect. Even when Ca(2+) was uncaged along the whole inner surface of the basolateral membrane, no Cl(-) current was elicited. There was little current deactivation at a high cytosolic Ca(2+) concentration ([Ca(2+)](c)), but at a low [Ca(2+)](c) there was clear voltage-dependent deactivation, which increased with hyperpolarization. Functional Ca(2+)-activated Cl(-) channels are expressed exclusively in the apical membrane and channel opening is strictly regulated by [Ca(2+)](c) and membrane potential. Ca(2+)-activated Cl(-) channels do not mediate Cl(-) uptake at the base, but acetylcholine-elicited local [Ca(2+)](c) spiking in the apical pole can regulate fluid secretion by controlling the opening of these channels in the apical membrane.

Animals↗
PubMed2004

Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells.

Elevations of cytoplasmic free calcium concentrations ([Ca(2+)](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl(-) through Ca(2+)-activated Cl(-) channels, while Cl(-) enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl(-) secretion, we analyzed carbachol (CCh)-activated Cl(-) currents in submandibular acinar cells using the "gramicidin-perforated patch recording configuration." Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl(-) currents in the gramicidin-perforated patch recording were carried by Cl(-) efflux via Cl(-) channels, dependent upon Cl(-) entry through Cl(-) transporters expressed in the acinar cells. CCh-evoked oscillatory Cl(-) currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl(-) currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO(3)(-) had significant effects, suggesting that the cotransporter rather than parallel operations of Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers is the primary Cl(-) uptake pathway. Pharmacological manipulation of the activities of the Ca(2+)-activated Cl(-) channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl(-) currents, while adjusting to the rate imposed by the Ca(2+)-activated Cl(-) channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl(-) movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Angiogenesis Inhibitors↗
PubMed1994

Carbonic anhydrase II expression in rat type II pneumocytes.

Pulmonary carbonic anhydrase (CA) activity plays important roles in carbon dioxide exchange, fluid secretion, and pH regulation. This study reports the use of molecular and immunologic techniques to characterize expression of the high-activity cytosolic isoenzyme CA II in rat lung tissue. Northern blot analysis of RNA isolated from various rat tissues revealed that the lung is a site of abundant tissue-specific CA II gene expression. The cell type primarily responsible for CA II expression in the lung was identified by immunohistochemistry as the alveolar type II pneumocyte. RNA blot and immunoblot analyses of isolated rat type II cells in culture confirmed CA II expression by this cell type. Little immunoreactive CA I and no CA IV was detected in these cells. Inhibition studies confirmed that the majority of CA activity in isolated type II cells is attributable to CA II. CA II expression was found to continue in these cells beyond 72 h in culture, a timeframe during which these cells had dedifferentiated. The ontologic pattern of CA II expression in the lung was found by RNA blot analysis to be disparate from that of the surfactant-associated proteins. These observations suggest roles for CA II in alveolar pneumocytes independent of (or in addition to) participation in surfactant biology. Such roles may include the regulation of fluid secretion or facilitation of carbon dioxide elimination.

Animals↗
PubMed1995

CIC-2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth.

Growth and differentiation of the fetal lung are dependent on chloride and fluid secretion, yet the specific molecular identities of fetal chloride channels have not been fully determined. In this study, we demonstrate mRNA expression of the volume-activated chloride channel, CIC-2, in fetal rat lung using reverse-transcriptase polymerase chain reaction (RT-PCR) and ribonuclease (RNase) protection assay. By RNase protection assay, CIC-2 mRNA expression is most abundant in fetal lung and diminishes after birth until it is almost undetectable in adult rat lung. To confirm this result at the protein level, a C-terminal fragment of CIC-2 cDNA derived from 19-day fetal rat lung was cloned into an expression plasmid. The truncated 33-kD CIC-2 protein was expressed in Escherichia coli and purified by column chromatography. Polyclonal antibodies to this antigen were raised in chickens, and the antisera detected a 94-kD protein in fetal rat lung homogenates by Western blotting. Protein expression of CIC-2 was most abundant in mid and late gestation and decreased significantly shortly after birth, as would be predicted by the RNase protection data. CIC-2 protein was localized along the apical surface of fetal airway epithelium by immunocytochemistry. The abundant fetal expression of CIC-2 RNA and protein supports the hypothesis that CIC-2 is important to fetal lung development, and its apical location suggests that it may be involved in fluid secretion during normal lung morphogenesis.

Animals↗
PubMed1993

Epithelial polarity and differentiation in polycystic kidney disease.

Renal cysts are central pathological features in a number of human congenital and acquired diseases, and produce significant morbidity and mortality. This review describes our laboratory's efforts to identify specific alterations in epithelial cell polarity and differentiation associated with renal tubular cyst formation and progressive enlargement. Studies in a murine model of human autosomal recessive polycystic kidney disease, the C57BL/6J cpk/cpk (CPK) mouse have demonstrated quantitative (increased activity) and qualitative (apical membrane distribution) alterations in Na+,K(+)-adenosine triphosphatase activity that mediate tubular cyst formation. Proximal tubular cyst formation in CPK kidneys is characterized by increased activity of a basolateral Na+,K(+)-ATPase, which drives organic anion secretion and consequent tubular fluid secretion. In contrast, collecting tubule cyst formation is characterized by increased apical membrane Na+,K(+)-ATPase expression, which may be a marker of the relatively undifferentiated phenotype of cyst lining cells. If such apically expressed enzyme is active, it may have pathogenic import in collecting tubule cyst formation and enlargement by mediating net basal to apical vectorial solute and fluid transport.

Animals↗
PubMed2003

Modulation of [Ca2+]i signaling dynamics and metabolism by perinuclear mitochondria in mouse parotid acinar cells.

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.

Adenosine Triphosphate↗
PubMed2004

Altered ATP-sensitive P2 receptor subtype expression in the Han:SPRD cy/+ rat, a model of autosomal dominant polycystic kidney disease.

The effects of extracellular ATP on fluid secretion and reabsorption by renal epithelial cells, as well as its known effects on cell proliferation and death, are potentially important contributory factors in the development and growth of renal cysts. In this study, we have investigated the protein and mRNA expression of several P2Y receptor subtypes (P2Y(1,2,4,6)), as well as the P2X(5) and P2X(7) receptors, in kidney tissue from the Han:SPRD (cy/+) rat model of polycystic kidney disease. All of the P2Y receptors tested for, and the P2X(5) and P2X(7) subtypes, were located on the cyst-lining cells of Han:SPRD (cy/+) rat polycystic kidneys; most immunostaining was cytosolic and we could not confidently localize it to one or other membrane. However, the staining pattern for P2Y(6) was uniquely granular when compared with the other P2 receptors. P2Y(2) and P2Y(6) receptor mRNA was increased in both homozygote (cy/cy) and heterozygote (cy/+) rat kidneys when compared with unaffected littermates. The protein levels of P2Y(2) and P2Y(6) receptors were also increased, being undetectable or at a low level, respectively, in control tissue. Finally, P2X(7) receptor mRNA was increased in cy/+, but not in cy/cy rat kidneys. Our results show that a number of P2Y receptor subtypes, as well as the P2X(5) and P2X(7) receptors, are clearly expressed in cyst-lining cells in the Han:SPRD (cy/+) rat model of renal cystic disease. Furthermore, P2Y(2) and P2Y(6) receptor mRNA and protein levels are markedly increased in cystic rat kidneys compared with normal rats of the same genetic background. Thus, the most consistent findings were an increase in the expression of P2Y(2), P2Y(6) and P2X(7) receptors in cystic tissue. Given the widely reported effects of stimulating these P2 receptor subtypes in epithelial and other renal cells, they could contribute to the development and growth of renal cysts: extracellular ATP and its products 'trapped' in cyst fluid may activate P2 receptors expressed by cyst-lining cells, causing cyst expansion from increased fluid secretion and/or reduced reabsorption, as well as an increase in cell turnover (re-modeling).

Adenosine Triphosphate↗