Intercellular communication mediated by the extracellular calcium-sensing receptor.

Agonist-evoked, intracellular Ca2+-signalling events are associated with active extrusion of Ca2+ across the plasma membrane, implying a local increase in Ca2+ concentration ([Ca2+]) at the extracellular face of the cell. The possibility that these external [Ca2+] changes may have specific physiological functions has received little consideration in the past. Here we show that, at physiological ambient [Ca2+], Ca2+ mobilization in one cell produces an extracellular signal that can be detected in nearby cells expressing the extracellular Ca2+-sensing receptor (CaR), a cell-surface receptor for divalent cations with a widespread tissue distribution. The CaR may therefore mediate a universal form of intercellular communication that allows cells to be informed of the Ca2+-signalling status of their neighbours.

Identification and localization of the extracellular calcium-sensing receptor in human breast.

The extracellular calcium (Ca2+o)-sensing receptor (CaR) plays a critical role in maintaining Ca2+o homeostasis in mammals by virtue of its presence in parathyroid gland and kidney. The breast is well recognized as a Ca(2+)-handling organ, and the effects of altering Ca2+o on the proliferation of breast epithelial cells are well documented. To date there are no data regarding the expression and localization of CaR in breast tissue. In the present study, we assessed the distribution of CaR messenger ribonucleic acid (mRNA) and protein in normal and fibrocystic human breast tissue as well as in ductal carcinoma of the breast using RT-PCR, Northern analysis, and immunohistochemistry with CaR-specific antisera. In all tissues, RT-PCR performed using sense and antisense primers based on the sequence of the human parathyroid CaR complementary DNA amplified a product of the size expected (425 bp) for genuine CaR transcripts. Nucleotide sequencing of RT-PCR products confirmed more than 99% homology with human parathyroid CaR complementary DNA. Although insufficient quantities of mRNA were isolated from normal and fibrocystic tissue for Northern analysis, a single 5.2-kb CaR transcript was expressed in malignant breast tissue similar to the major CaR transcript in human parathyroid. Localization of CaR protein by immunohistochemistry showed specific CaR staining of the ductal epithelial cells of the breast in all three tissue types. These findings indicate the presence of CaR mRNA and protein in the breast, providing indirect evidence that the CaR may have some role(s) in the control of Ca2+ transport, epithelial cell proliferation, and/or other processes in normal and abnormal breast tissue.

Effect of 16,16-dimethyl prostaglandin E2 on gastric epithelial cell membrane potentials and resistances.

Intracellular microelectrode techniques were employed to examine the effects of 16,16-dimethyl prostaglandin E2 (dmPGE2) on Necturus antral mucosa epithelial cell membrane potentials and resistances. Necturus antral mucosa was mounted in a modified Ussing chamber and stable intracellular impalements were obtained. Addition of 0.01 microgram/ml dmPGE2 to the mucosal solution produced an increase of the apical cell membrane potential (Vmc) from -44.3 +/- 3.1 to -53.5 +/- 3.8 mV (p less than 0.001) and of the basolateral cell membrane potential (Vcs) from -48.8 +/- 2.8 to -57.7 +/- 3.2 mV (p less than 0.001). This reversible, dose-dependent hyperpolarization of both cell membranes was accompanied by a decrease in the electrical resistance of the apical membranes (Ra) from 2550 +/- 250 omega/cm2 to 1870 +/- 210 omega/cm2 (p less than 0.05) and a decrease in the resistance of the basolateral membrane (Rb) from 1020 +/- 250 omega/cm2 to 630 +/- 80 omega/cm2 (p less than 0.05). In addition, there was an increase in the resistance of the shunt (intercellular junction, Rs), the major route of transepithelial ion flow, from 710 +/- 60 omega/cm2 to 750 +/- 80 omega/cm2 (p less than 0.05). Thus dmPGE2 increased the cell membrane potentials and reduced the ionic permeability of the intercellular junction.

Comparison of gastric mucosal blood flow as measured by H2 gas clearance and microspheres during secretory stimulation and inhibition.

H2 clearance is a recently described method of measuring gastric mucosal blood flow that has great potential for clinical use. However, the effects of luminal pH and of secretory activity of the gastric mucosa on the accuracy of H2 clearance measurements have not been systematically examined. We therefore tested the validity of H2 clearance measurements at different pHs in both in vitro and in vivo systems. In addition, we compared measurements by H2 clearance and radioactive microspheres during stimulation and suppression of acid secretion. In vitro, H2 washout was relatively constant over a range of pHs from 2.0 to 8.0. In chambered segments of canine fundus in vivo, H2 clearance was not significantly affected by pH of the luminal solutions either in the resting state or at lower blood flows induced during infusion of vasopressin. Finally, there was a close correlation (r = 0.85; p less than 0.001) between H2 clearance and microsphere measurements under resting conditions, during intravenous histamine stimulation, and after infusion of cimetidine to suppress acid secretion. In summary, H2 clearance reliably and accurately measures gastric mucosal blood flow at different luminal pHs and under conditions that stimulate or suppress acid secretion.

Pentagastrin selectively modulates levels of mRNAs encoding apical H/K adenosine triphosphatase and basolateral Na-K-Cl cotransporter in rat gastric fundic mucosa.

BACKGROUND: Gastrin regulates gastric acid secretion and gastric mucosal cell proliferation. We hypothesized that pentagastrin administration would affect mRNA levels of two membrane proteins that are important during stimulated states of HCl secretion, the basolateral Na-K-Cl cotransporter (BSC) and the apical H/K adenosine triphosphatase (H/K). METHODS: Two groups of Fischer rats received intraperitoneal injections of pentagastrin (2.5 or 25 micrograms/kg) every 8 hours for three doses. A third group served as controls. An additional group received pentagastrin plus the gastrin receptor antagonist (GRA) L740,093. Fundic mucosae were subjected to semiquantitative Northern analysis of mRNAs encoding H/K and BSC. The mRNA for Na/K adenosine triphosphatase (Na/K), a transport protein not involved directly in acid secretion, also was evaluated. RESULTS: Administration of pentagastrin caused dose-dependent increases in levels of mRNAs encoding H/K and BSC but had no significant effect on levels of Na/K mRNA. Administration of GRA prevented the pentagastrin-induced changes in mRNA levels for these transporters. CONCLUSIONS: Pentagastrin administration selectively up-regulates levels of mRNA encoding membrane proteins involved in acid secretion. The up-regulation of the mRNAs encoding BSC during pentagastrin stimulation indicates that regulation of basolateral Cl- movement may be as important as the regulation of apical H+ movement under stimulated states.

Intracellular pH (pHi) in gastric surface epithelium is more susceptible to serosal than mucosal acidification.

Intracellular microelectrode techniques were used to examine the effects of mucosal or serosal acidification on intracellular pH (pHi) in gastric surface epithelial cells. Necturus antrum was mounted in a modified Ussing chamber, and pHi was determined from the difference between the potentials recorded by intracellular conventional and pH-sensitive microelectrodes. In tissues bathed with bicarbonate-buffered Ringer's solution (pH 7), acidification of the mucosal solution to pH 4.5 by isotonic replacement of the NaHCO3 with NaCl had no significant effects on pHi. In contrast, acidification of the serosal solution to pH 4.5 by replacing the bicarbonate reduced pHi from 7.32 +/- 0.04 to 6.95 +/- 0.06 (p less than 0.001, n = 8). Similarly, in tissues bathed with HEPES-buffered Ringer's solution (pH 7.0), pHi was unaffected by reducing the mucosal solution pH to 4.5 with HCl but fell 0.21 +/- 0.05 pH units (p less than 0.01, n = 7) during acidification of the serosal solution to pH 6. These results suggest that gastric epithelium is more sensitive to acidification from the serosal than the mucosal side. Such a finding is consistent with the concept of a gastric mucosal barrier to luminal acid. It may also explain the gastric epithelium's greater sensitivity to acute ulceration during systemic acidosis.

Regional gastric mucosal blood flow after parietal cell vagotomy in dogs.

In this study we used the recently validated H2 clearance method to perform endoscopic measurements of gastric mucosal blood flow (MBF) in anesthetized dogs before and after parietal cell vagotomy (PCV). Under resting conditions, MBF in the gastric corpus before PCV was 72 +/- 5 ml/min/100 gm. This was not altered significantly at 4, 8, or 16 weeks after PCV, and there were not significant long-term changes in MBF on the greater or lesser curvatures of the corpus individually. Before PCV infusion of pentagastrin (8 micrograms/kg/hr) elicited increases in corpus MBF to 104 +/- 4 ml/min/100 gm, accompanied by increases in gastric acid output from resting levels of 2.1 +/- 0.9 to 38.6 +/- 2.4 mEq/hr (p less than 0.001). PCV significantly reduced pentagastrin-stimulated acid secretion by 50%, and secretory inhibition was accompanied by significant reductions in pentagastrin-stimulated MBF in the corpus. Pentagastrin did not alter antral MBFs before or after PCV. In summary PCV does not elicit significant long-term changes in resting MBF in different regions of the gastric corpus, and PCV significantly diminishes increases in acid output and corpus MBF that are normally stimulated by pentagastrin. These observations suggest that alterations in gastric MBF after PCV may be attributable to alterations in acid secretion.

Gastrin-mediated effects of omeprazole on rat colon mucosa.

BACKGROUND: Omeprazole increases circulating gastrin levels, which in turn may affect the growth and differentiation of colon mucosa. Chloride transport mechanisms in normal colon were analyzed as markers for possible trophic actions of endogenous hypergastrinemia. METHODS: Four groups of Fischer rats were studied for 10 days. Group 1 (baseline) received no treatment. Group 2 received omeprazole only. Group 3 received omeprazole plus vehicle. Group 4 received omeprazole plus CCK-B gastrin receptor antagonist (GRA) L740,093 in vehicle. On day 10 serum gastrin was assayed. Colon mucosa was analyzed for protein and DNA content. Semiquantitative Northern analysis measured levels of messenger RNA (mRNA) encoding for key Cl- transporters: Na-K-Cl cotransporter (Cl- secretion in crypts), Cl-/HCO3- exchanger (Cl- absorption in villi), and Na/K adenosine triphosphatase (not directly involved in Cl- transport). RESULTS: Omeprazole increased gastrin levels, which were not altered by vehicle or GRA. Omeprazole increased protein, DNA, and Na/K adenosine triphosphatase mRNA levels, with no effect by GRA. In contrast, omeprazole decreased Na-K-Cl and Cl-/HCO3- mRNA levels, effects that were partly reversed by GRA. CONCLUSIONS: Omeprazole augments growth index values of colon mucosa independent of serum gastrin. Against a background of omeprazole-induced achlorhydria hypergastrinemia appears to influence differentiation rather than growth of normal colon mucosa.

Broncholithiasis and thoracoabdominal actinomycosis from dropped gallstones.

We report a case of successfully managed invasive, thoracoabdominal actinomycosis caused by the intraperitoneal spillage of gallstones during laparoscopic cholecystectomy. The infected gallstones traversed the diaphragm, migrated into the lung parenchyma, and obstructed a segmental bronchus, causing pneumonia. Treatment involved retrieval of the obstructing stone, debridement and drainage of the pleuroperitoneal phlegmon/abscess, and intravenous antibiotics. The case illustrates the need to remove gallstones at the time of cholecystectomy.

Symptomatic gallstones in patients with spinal cord injury.

Patients with spinal cord injury (SCI) have an increased prevalence of cholelithiasis. The goal of this study was to clarify the presentation and management of symptomatic gallstone disease in patients with SCI. We performed a retrospective study of presentation of gallstone complications in patients with SCI who underwent cholecystectomy for complications of gallstone disease. The West Roxbury Veterans Administration Medical Center SCI registry (605 patients) was searched for patients who had undergone cholecystectomy more than 1 year after SCI (35 patients). Gallbladder disease profiles for the 35 patients undergoing cholecystectomy for complications of gallstone disease were prepared, including demographics, clinical presentation, diagnostic studies, operative and pathologic findings, and postoperative complications. All patients were white. Thirty-four were male and the mean age was 50 years (range 35 to 65 years). The majority of patients (66%) complained of right upper quadrant abdominal pain, even those patients with SCI at high (i.e., cervical) levels. Of the 35 patients in our study group, 22 (63%) had biliary colic and chronic cholecystitis, nine (26%) had acute cholecystitis (gangrenous cholecystitis in two), two (6%) had choledocholithiasis symptoms or cholangitis, and two (6%) had gallstone pancreatitis. Major perioperative morbidity occurred in two (6%) of the 35 patients (pulmonary embolus; intraoperative hemorrhage), and there were no deaths. In the great majority of patients with SCI, cholelithiasis presents with chronic pain and not with life-threatening complications. Our findings suggest that presentation is no more acute in patients with SCI than in the general population. Characteristic symptoms and signs are not necessarily obscured by SCI injury, regardless of the level.

Selective increase in gastric mucosal mRNA encoding basolateral Na-K-2C1 cotransporter following ileostomy in the rat.

Results of previous studies suggest that major surgical resections or reconstructions of the distal small intestine can alter morphologic and functional properties of the stomach. Little is known about the effect of lesser surgical alterations such as construction of an ileostomy, on the morphology and transport properties of the gastric mucosa. To evaluate the effects of ileostomy, Sprague-Dawley rats underwent sham laparotomy (n = 10) or loop ileostomy construction (n = 10). After body weights had stabilized ( approximately 21 days) the animals were killed. Gastric mucosal scrapings were prepared for Northern blot analysis of messenger RNA levels for (1) H/K ATPase, found in parietal cells; (2) Na-K-2C1 cotransporter, found in both parietal and surface cells; and (3)Na/K ATPase, found in all gastric mucosal cells. Gastric mucosa from ileostomy animals was visibly hypertrophied compared to sham-operated animals. There was a 145% increase in the mRNA levels of the Na-K-2Cl cotransporter in gastric mucosa of the ileostomy group but no significant changes in H/K ATPase or Na/K ATPase mRNA levels. Construction of an ileostomy selectively enhances expression of the Na-K-C1 cotransporter in the gastric mucosa. Further studies are required to understand the neurohumoral stimuli underlying this selective response.

Selective decreases in levels of mRNA encoding a water channel (AQP3) in ileal mucosa after ileostomy in the rat.

Water channels (aquaporins) provide pathways for water permeation in a variety of epithelia. Aquaporin-3 (AQP3) has been localized to the basolateral membranes of epithelial cells in the small intestine, but mechanisms that regulate its expression and function have not been explored. To determine whether luminal content may influence intestinal AQP3 gene expression, adult Sprague-Dawley rats underwent sham laparotomy (N = 11) or loop ileostomy (N = 9) and were killed 8 days after procedures. Northern blot analysis was used to measure messenger RNA (mRNA) levels for AQP3 and the Na(+)/K(+) ATPase, a housekeeping transporter that regulates cellular levels of Na(+) and K(+). At sacrifice, histologic examination revealed only minimal changes in mucosal morphology. In sham animals, Na/K mRNA levels increased moderately in distal regions of the small intestine. Ileostomy did not alter these levels in any region. In contrast, in sham animals, AQP3 mRNA levels increased along the length of the intestine and were markedly higher in the distal ileum. Diversion of luminal contents decreased AQP3 mRNA levels in the postileostomy region by 30% to 50%. These findings indicate regional variations in expression of the AQP3 water channel in mucosa of the small intestine. In addition, they suggest that AQP3 gene expression may depend on the presence of luminal contents.

Luminal osmolarity downregulates gene expression of Na+/H+ exchanger (NHE3) in rat colon mucosa.

Water-coupled Na&sup+ absorption in the colon is mediated principally by Na+/H+ exchange (isoforms NHE2 and NHE3). To determine whether luminal ion composition or osmolarity influences NHE expression in colon mucosa, two groups (n = 6 in each) of adult male Sprague-Dawley rats underwent sham laparotomy or loop ileostomy. In these studies, diversion did not markedly alter mRNA levels for NHE2, NHE3, or Na+/K+, at 8 or 21 days, indicating that loss of luminal volume does not alter NHE gene expression. To evaluate the effects of specific luminal components, we infused equal volumes of half-normal (154 mOsm) or iso-osmolar (308 mOsm) solutions of saline and mannitol into the diverted colon. All solutions elicited significant (45% to 60%; P <0.05) decreases in mRNA levels for NHE3, with iso-osmolar mannitol eliciting the greatest changes. Decreases in NHE2 and Na+/K+ mRNA levels were observed following these infusions but were not as marked as the changes for NHE3. These findings suggest that (1) loss of luminal Na+ is not, in itself, a signal that regulates NHE expression and (2) infusion of any solute, including Na+ itself, provides a signal to downregulate expression of NHE3 in colon mucosa.

Implications of sustained suppression of gastric acid secretion.

The implications of profound and sustained suppression of acid secretion are of increasing concern. Short-term inhibition of acid secretion by H2-receptor blockade or proton pump inhibition alters the gastric luminal flora and increases the risk of nosocomial pneumonia in critically ill patients who are receiving prophylaxis for stress gastritis. Long-term suppression alters gut flora, carcinogen levels in the gastric lumen, and the hormonal milieu, leading to proliferative changes in the fundic mucosa. Previous reports have noted a significant incidence of gastric malignancies in the achlorhydric environment of atrophic gastritis and pernicious anemia. Concern has also been expressed regarding the possibility of gastric neoplasia that arises after vagotomy and distal gastrectomy. The exact risk of gastric epithelial and endocrine hyperplasia or neoplasia in patients receiving potent antisecretory agents is not yet known, but such risks cannot be dismissed until long-term follow-up studies are available. The relationship between sustained suppression of acid secretion and the proliferation of epithelial and endocrine elements may provide insight into processes that regulate replication and growth of cells in the gastric mucosa.

Effects of platelet-activating factor on canine gastric mucosal blood flow and permeability to luminal acid.

Recent studies suggest that platelet-activating factor, a newly described phospholipid released during acute inflammation, may predispose the gastric mucosa to acute ulceration. This study was performed to evaluate whether this substance might contribute to acute injury through its effects on mucosal microcirculation or by increasing permeability of the gastric mucosa to luminal acid. Platelet-activating factor was infused at a rate of 1 microgram/min directly into the artery supplying a chambered segment of canine gastric corpus and both significantly decreased venous outflow and mucosal blood flow and enhanced the efflux of volume, sodium ions, and potassium ions into the lumen. Thus, as in other capillary beds, platelet-activating factor appeared to induce stasis of flow and local accumulation of interstitial fluids which then leaked into the lumen. Platelet-activating factor did not significantly alter the back-diffusion of hydrogen ions into the mucosa. Thus, platelet-activating factor may not directly influence mucosal permeability to luminal acid, and it may predispose the gastric mucosa to acute injury through its effects on the microvasculature and other aspects of the inflammatory process.

Protective effect of exogenous phospholipid on aspirin-induced gastric mucosal injury.

The protective effects of exogenous phospholipid on aspirin-induced gastric mucosal injury were examined in a canine chamber model which provided two separate segments of mucosa supplied by a single vascular pedicle. In each dog, one segment was treated with a suspension of surface-active phospholipid, similar in composition to that normally present in the gastric mucosa, whereas the other segment served as the control. Pretreatment of the test segments significantly prevented aspirin-induced disruption of the mucosal barrier as evidenced by an increase in potential difference and a decrease in acid back-diffusion and sodium ion and potassium ion flux. These findings were associated with a marked reduction in the degree of mucosal injury. Our results support the recent hypothesis that surface-active phospholipid plays an important role in gastric mucosal defense against the damaging effects to luminal acid.

Effects of aspirin and acetic acid on intracellular pH in necturus gastric mucosa.

Intracellular microelectrode techniques were employed to examine the effects of luminal aspirin and acetic acid on intracellular pH and cell membrane potential in the surface epithelial cells of Necturus antrum. Antral mucosa was mounted in a modified Ussing chamber, and intracellular pH was determined from the difference between the potentials recorded by intracellular conventional and pH-sensitive microelectrodes. Under neutral conditions (pH7), aspirin (5 mM) hyperpolarized (-7.5 +/- 1 mV, p less than 0.0001) and acetic acid (5 mM) depolarized (+4 +/- 0.08 mV, p less than 0.001) cell membrane potential. Neither agent had any significant effect on intracellular pH. Under acidic conditions (pH 4.5), aspirin (5 mM) reduced the intracellular pH from 6.99 +/- 0.03 to 6.87 +/- 0.04 (p less than 0.001) and depolarized cell membrane potential from -36.7 +/- 1.5 to -30.3 +/- 1.6 mV, p less than 0.001). Similarly, acetic acid (5 mM) acidified the cells (-0.20 +/- 0.02, p less than 0.001) and depolarized cell membrane potential (+9.6 +/- 1.9 mV, p less than 0.01). These changes suggest that, in the absence of luminal acid, small organic acids, such as aspirin and acetic acid, may have complex effects on the ionic conductances of the surface cell membranes without altering intracellular pH. In contrast, under acidic conditions, these agents increase the permeability of the apical cell membrane-to-acid back-diffusion from the gastric lumen.

Colon and rectal carcinoma.

In the United States the incidence of carcinoma of the colon or rectum appears to be increasing. Although certain dietary habits appear to be associated with disease incidence, the putative carcinogens in the lumen of the bowel remain unidentified. The use of clinical screening based on a combination of proctosigmoidoscopy and tests for occult fecal blood allows detection of colorectal carcinomas at an early stage; however, it is unclear whether such screening is cost-effective in persons over 40 years of age or if treatment undertaken on the basis of screening results truly alters the natural history of the disease. Surgical resection is the mainstay of curative therapy, and its effectiveness will probably be enhanced by adjuvant x-ray therapy and chemotherapy.

Effect of growth hormone on intestinal Na+/glucose cotransporter activity.

BACKGROUND: Growth hormone (GH) has been used alone or as part of a defined regimen in the treatment of patients with short bowel syndrome; however its mode of action remains unclear. Growth hormone has been shown to increase amino acid, water, and electrolyte absorption from the small intestine. The acute effect of growth hormone on intestinal sugar transport has not been described previously. METHODS: Mucosal preparations of rat jejunum were mounted in the Ussing chamber. Growth hormone (2 x 10(-6) M or 8 x 10(-6) M) or vehicle was added to the serosal chamber 1, 3, or 5 hours later. Twenty or 40 minutes after growth hormone addition, 30 mmol/L 3-O-methylglucose was added to both chambers, and the change in short-circuit current (deltaIsc) was recorded. In separate experiments, tissues were pretreated with phloridzin, an inhibitor of Na+/glucose cotransport, before the addition of 3-O-methylglucose. In the final set of experiments, kinetic studies were performed. RESULTS: GH did not induce any alterations in baseline electrical parameters. Only tissues left in the chambers for 5 hours, but not 1 or 3 hours, before GH treatment displayed a greater 3-O-methylglucose-induced deltaIsc than controls (p < .05). The increase in Isc induced by 3-O-methylglucose was 100% phloridzin-inhibitable. Kinetic analysis showed that growth hormone administration is associated with an increase in Na+/glucose cotransporter maximal velocity (Vmax) but no significant change in carrier affinity for substrate (Km). CONCLUSIONS: Growth hormone increases intestinal sugar transport, but only in tissue that has not been exposed to endogenous GH for over 3 hours.