Modulation of single-nephron GFR in the db/db mouse model of type 2 diabetes mellitus. II. Effects of renal mass reduction

This study examines for the first time the effects of uninephrectomy (Nx) on modulation of whole kidney glomerular filtration rate (GFR), single-nephron GFR (SNGFR), and progression of diabetic nephropathy in the db/db mouse model of type 2 diabetes mellitus. To characterize SNGFR and tubuloglomerular feedback (TGF) responses to Nx and chronic neuronal nitric oxide synthase inhibition in the db/db mouse, we studied the effects of Nx on whole kidney GFR, SNGFR, and TGF characteristics in db/db and wild-type (WT) mice after Nx or sham Nx. We also documented progression of glomerular changes over a 6-mo period. Whole kidney GFR and SNGFR were significantly higher in db/db Nx than db/db sham mice, without change in proximal tubule reabsorptive rates. The TGF responses, determined as proximal-distal SNGFR differences, were brisk: 12.1 +/- 1.0 vs. 8.4 +/- 0.6 nl/min in WT sham (P < 0.05), 15.7 +/- 1.0 vs. 12.0 +/- 1.0 nl/min in WT Nx (P < 0.05), and 17.8 +/- 1.3 vs. 14.3 +/- 1.0 nl/min in db/db Nx (P < 0.05) mice. Chronic ingestion of the neuronal nitric oxide synthase inhibitor S-methylthiocitrulline for 2-3 wk after Nx had no effect on SNGFR or the TGF response. These studies show further elevations in whole kidney GFR and SNGFR in these hyperglycemic morbidly obese db/db mice, with an intact TGF system after Nx. In addition, in the db/db Nx mice, 4-6 mo after Nx, there was an exacerbation of the lesions of diabetic nephropathy, as quantified by a significant increase in the ratio of mesangial surface area to total glomerular surface area.     

p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats

Angiotensin II (ANG II) infusion increases renal superoxide (O(2)(-)) and enhances renal vasoconstriction via macula densa (MD) regulation of tubuloglomerular feedback, but the mechanism is unclear. We targeted the p22(phox) subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) with small-interfering RNA (siRNA) to reduce NADPH oxidase activity and blood pressure response to ANG II in rats. We compared single nephron glomerular filtration rate (SNGFR) in samples collected from the proximal tubule (PT), which interrupts delivery to the MD, and from the distal tubule (DT), which maintains delivery to the MD, to assess MD regulation of GFR. SNGFR was measured in control and ANG II-infused rats (200 ng.kg(-1).min(-1) for 7 days) 2 days after intravenous injection of vehicle or siRNA directed to p22(phox) to test the hypothesis that p22(phox) mediates MD regulation of SNGFR during ANG II. The regulation of SNGFR by MD, determined by PT SNGFR-DT SNGFR, was not altered by siRNA in control rats (control + vehicle, 13 +/- 1, n = 8; control + siRNA, 12 +/- 2 nl/min, n = 8; not significant) but was reduced by siRNA in ANG II-treated rats (ANG II + vehicle, 13 +/- 2, n = 7; ANG II + siRNA, 7 +/- 1 nl/min, n = 8; P < 0.05). We conclude that p22(phox) and NADPH oxidase regulate the SNGFR during ANG II infusion via MD-dependent mechanisms.     

The effect of secretin on acid and pepsin secretion and gastrin release in the totally isolated vascularly perfused rat stomach

The effect of secretin on acid and pepsin secretion and gastrin release in the totally isolated vascularly perfused rat stomach was studied. With the phosphodiesterase inhibitor isobutyl methylxanthine (IMX) added to the vascular perfusate, baseline acid secretion was 4.7 +/- 1.1 (mean +/- S.E.M.) mumol/h and baseline pepsin output 1147 +/- 223 micrograms/h. Secretin significantly inhibited acid output to a minimum of 1.4 +/- 0.2 mumol/h at a concentration of 25 pM in the vascular perfusate (P less than 0.01). Pepsin output was not significantly different from baseline at any of the secretin doses tested. Threshold secretin concentration for acid inhibition was 5 pM. IMX stimulated gastrin output from 48 +/- 9 pM in the basal state to 95 +/- 13 pM after IMX (P less than 0.01). Secretin inhibited gastrin release only at the maximal dose of 625 pM, when gastrin concentration in the venous effluent decreased from 93 +/- 19 to 68 +/- 19 pM after secretin. Thus, in the totally isolated vascularly perfused rat stomach secretin in physiological concentrations inhibits acid secretion by a direct action on the acid secretory process and not via gastrin inhibition. The study also suggests that gastrin release at least in part is mediated via increased intracellular cAMP.     

Effect of acute hyperglycemia on basal, secretin and secretin + cholecystokinin stimulated exocrine pancreatic secretion in humans

Pancreatico-biliary secretion is reduced during acute hyperglycemia. We investigated whether alterations in pancreatico-biliary flow or volume output are responsible for the observed reduction in duodenal output of pancreatic enzymes and bilirubin during hyperglycemia. Eight healthy subjects were studied on two occasions during normoglycemia and hyperglycemia (15 mmol/l). Pancreatico-biliary output was measured by aspiration using a recovery marker under basal conditions (60 min), during secretin infusion (0.1 CU/kg.h) for 60 min and during secretin + CCK (0.5 IDU/kg.h) infusion for 60 min. Secretin was infused to stimulate pancreatico-biliary flow and volume output. Secretin significantly (P<0.005-P<0.05) increased volume and bicarbonate output and CCK significantly (P<0.01) increased the output of bilirubin, pancreatic enzymes, bicarbonate and volume, both during normoglycemia and hyperglycemia. During hyperglycemia basal, secretin stimulated and secretin + CCK stimulated total pancreatico-biliary output were significantly (P<0.005-P<0.05) reduced compared to normoglycemia. The incremental outputs, however, were not significantly different between hyper- and normoglycemia. Pancreatic volume output was significantly (P<0.05) reduced during hyperglycemia compared to normoglycemia under basal conditions (31+/-16 m/h versus 132+/-33 m/h) during secretin infusion (130+/-17 ml/h versus 200+/-34 m/h) and during secretin + CCK infusion (370+/-39 ml/h versus 573+/-82 ml/h). Plasma PP levels were significantly (P<0.05) reduced during hyperglycemia. It is concluded that 1) hyperglycemia significantly reduces basal pancreatico-biliary output 2) the incremental pancreaticobiliary output in response to secretin or secretin + CCK infusion is not significantly affected during hyperglycemia, 3) a reduction in volume output contributes to the inhibitory effect of hyperglycemia on pancreatico-biliary secretion, 4) hyperglycemia reduces PP secretion suggesting vagal-cholinergic inhibition of pancreatico-biliary secretion and volume during hyperglycemia.     

Modulation of single-nephron GFR in the db/db mouse model of type 2 diabetes mellitus

Hyperfiltration has been implicated in the progression toward diabetic nephropathy in type 2 diabetes mellitus (DM2). This study focuses for the first time on the in vivo modulation of single-nephron GFR (SNGFR) in the classic B6.Cg-m(+/+)Lepr(db)/J (db/db) mouse model of DM2. To obtain stable preparations, it was necessary to use a sustaining infusion of 3.3 ml.100 g body wt(-1) x h(-1), or higher. SNGFR (measured both proximally and distally) was greater in db/db vs. heterozygote (db/m) mice (P < 0.05) but not vs. the wild-type (WT) mice. The tubuloglomerular feedback (TGF) responses, determined as free-flow proximal vs. distal SNGFR differences, were significant in db/db mice (11.6 +/- 0.8 vs. 9.3 +/- 1.0 nl/min, P < 0.01), in db/m mice (8.0 +/- 0.8 vs. 7.2 +/- 0.6 nl/min, P < 0.02), and WT mice (9.9 +/- 0.6 vs. 8.9 +/- 0.7 nl/min, P < 0.05). After increasing the sustaining infusion in the db/db mice, to offset glycosuric urine losses, the SNGFR increased significantly, and the TGF response was abolished. In these volume-replete db/db mice, absolute fluid reabsorption measured both at the late proximal and distal tubular sites were significantly increased vs. db/m mice infused at 3.3 ml.100 g body wt(-1) x h(-1). After infusion of the neuronal nitric oxide synthase (nNOS) inhibitor S-methylthiocitrulline, SNGFR fell in both db/db and db/m mice. These studies show that SNGFR is elevated in this mouse model of DM2, is suppressed by nNOS inhibition, and is modulated by TGF influences that are altered by the diabetic state and responsive to changes in extracellular fluid volume.     

Effect of gastrointestinal hormones on choleresis from the isolated perfused rat liver

Using isolated perfused rat liver, the direct effect of secretin, glucagon, caerulein, insulin and somatostatin on choleresis was investigated. When the liver was perfused in the absence of sodium taurocholate, the bile volumes were: control, 0.33 +/- 0.01 (mean +/- S.E.M.) ml/10 g liver per 50 min; secretin 0.05 U/ml, 0.39 +/- 0.01 (P less than 0.01); glucagon 10(-10) M, 0.44 +/- 0.02 (P less than 0.01); caerulein 10(-8) M, 0.34 +/- 0.03 (n.s.); insulin 1 mU/ml, 0.35 +/- 0.02 (n.s.); glucagon plus somatostatin 10(-7) M, 0.46 +/- 0.03 (n.s. vs. glucagon alone), respectively. When 10(-5) M sodium taurocholate was present in the perfusate, the bile volumes were: control, 0.61 +/- 0.03; secretin, 0.63 +/- 0.01 (n.s.); glucagon, 0.70 +/- 0.01 (P less than 0.05); caerulein, 0.55 +/- 0.01 (n.s.); insulin, 0.62 +/- 0.04 (n.s.); somatostatin, 0.59 +/- 0.01 (n.s.); respectively. Glucagon increased glucose output and cyclic AMP in the effluent from the liver neither of which were suppressed by somatostatin. Secretin increased cyclic AMP but not glucose output. These results indicate that glucagon has the most potent action on bile acid-independent canalicular bile, that caerulein and insulin do not act on canalicular bile production directly and that somatostatin does not directly suppress canalicular bile production nor hepatic glucose output produced by glucagon in rats.     

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

Bicarbonate-water interactions in the rat proximal convoluted tubule. An effect of volume flux on active proton secretion

The effect of volume absorption on bicarbonate absorption was examined in the in vivo perfused rat proximal convoluted tubule. Volume absorption was inhibited by isosmotic replacement of luminal NaCl with raffinose. In tubules perfused with 25 mM bicarbonate, as raffinose was increased from 0 to 55 to 63 mM, volume absorption decreased from 2.18 +/- 0.10 to 0.30 +/- 0.18 to -0.66 +/- 0.30 nl/mm X min, respectively, and bicarbonate absorption decreased from 131 +/- 5 to 106 +/- 8 to 91 +/- 13 pmol/mm X min, respectively. This bicarbonate-water interaction could not be attributed to dilutional changes in luminal or peritubular bulk phase bicarbonate concentrations. Inhibition of active proton secretion by acetazolamide abolished the effect of volume flow on bicarbonate absorption, which implies that the bicarbonate reflection coefficient is close to 1 and eliminates the possibility of solvent drag across the tight junction. When the luminal bicarbonate concentration was varied, the magnitude of the bicarbonate-water interaction increased with increasing luminal bicarbonate concentration. The largest interaction occurred at high luminal bicarbonate concentrations, where the rate of proton secretion has been previously shown to be independent of luminal bicarbonate concentration and pH. The results thus suggest that a peritubular and/or cellular compartment exists that limits bicarbonate diffusion, and where pH changes secondary to bicarbonate-water interactions (solute polarization) alter the rate of active proton secretion.     

The effects of atrial natriuretic peptide on whole-kidney and proximal straight tubular function in the rabbit

The mechanisms by which atrial natriuretic peptide (ANP) produces a diuresis and natriuresis remain unclear. It has been suggested that the major if not sole mediator of ANP's renal effects is a hemodynamically induced increase in glomerular filtration rate (GFR). Data from clearance studies in anesthetized rabbits demonstrate that ANP administration can produce a significant increase in absolute and percentage sodium excretion (42.0 +/- 5.9----64.6 +/- 10.2 mu eq/min, P less than 0.01, and 1.97 +/- 0.28----3.12 +/- 0.35%, P less than 0.001, respectively) without increasing GFR (16.8 +/- 2.1----16.1 +/- 2.5 cc/min, P greater than 0.30). The natriuresis occurred despite a fall in renal plasma flow (RPF) (56.7 +/- 7.0----44.5 +/- 9.4 cc/min, P less than 0.01), a rise in filtration fraction (0.33 +/- 0.01----0.46 +/- 0.05, P less than 0.01), and an unchanged filtered load of sodium (2.28 +/- 0.27----2.16 +/- 0.32 mu eq/min, P greater than 0.10). Isolated tubular microperfusion studies demonstrated that ANP, present as a 10(-9) M concentration in the solution bathing perfused proximal straight tubules (PST), did not affect fluid flux (Jv) (0.38 +/- 0.07----0.41 +/- 0.07 nl/mm/min, P greater than 0.30) or phosphate reabsorption (Jp) (1.50 +/- 0.5----1.38 +/- 0.36 pmole/mm/min, P greater than 0.50). When ANP was infused into rabbits prior to harvesting the PSTs for isolated tubular microperfusion and the results were compared to tubules taken from control animals, there was again no effect on Jv (0.37 +/- 0.05 vs 0.42 +/- 0.05 nl/mm/min, P greater than 0.50) or Jp (2.41 +/- 0.27 vs 2.42 +/- 0.44 pmole/mm/min, P greater than 0.90). These findings suggest that ANP can inhibit sodium transport without increasing whole-kidney GFR or RPF, but does not directly inhibit transport in the proximal straight tubule.     

The effect of neurotensin and secretin on gastric acid secretion and mucosal blood flow in man

Neurotensin stimulates pancreatic secretion directly and by potentiating the effect of secretin. Neurotensin also inhibits gastric secretion. Secretin inhibits gastric secretion as well, but whether it also interacts with neurotensin is not known. Secretin is known to inhibit gastric mucosal blood flow (GMBF). The effect of neurotensin on GMBF is not known. Acid secretion (triple lumen perfused orogastric tube) and GMBF ([14C]aminopyrine clearance) were therefore measured in 6 subjects during neurotensin, secretin and neurotensin plus secretin infusions. Neurotensin plus secretin reduced acid secretion by a median 130 (range 34-394) mumol/min which was significantly greater than either neurotensin at 36 (7-67) mumol/min or secretin 54 (20-347) mumol/min alone (P less than 0.05). This effect appeared independent of GMBF. Neurotensin plus secretin reduced GMBF by 14 (12-27) ml/min but not significantly more than neurotensin at 11 (3-20) ml/min or secretin 18 (2-27) ml/min alone. Further, there was no correlation between changes in acid output and GMBF during infusion of the peptides. We conclude that the inhibitory effects of neurotensin and secretin on gastric secretion are at least additive and together they may function as an 'enterogastrone'.     

脑室内注射高张盐水抑制近曲小管对水和钠的重吸收

实验在麻醉大鼠上进行。用锂清除率为指标观察脑室内注射高张盐水对近曲小管重吸收水和钠的影响。在切断单侧肾神经的动物中,脑室内注射高张盐水后的锂清除率与肾小球滤过率比值在去神经侧肾脏从0.37±0.04增加至0.51±0.05(P<0.01);神经完好侧肾脏则从0.26±0.03增加至0.31±0.04(P<0.05);双侧肾脏的肾小球滤过率、尿量、尿钠和尿钾量均增加,且去肾神经肾脏的增加幅度高于肾神经完好肾脏。在肾小管微穿刺实验中,脑室内注射高张盐水后,近曲小管末段小管液流量从24.42±1.84nl/min增加至31.86±3.09nl/min(P<0.01),小管液的渗透压无显著变化。以上实验结果表明,脑室内注射高张盐水引起的利尿、尿钠增多反应与肾小球滤过率增加和近曲小管对水、钠重吸收减少有关,体液因素在该反应中可能起主要作用。     

Acute saline expansion increases nephron filtration and distal flow rate but maintains tubuloglomerular feedback responsiveness: role of adenosine A(1) receptors

Temporal adaptation of tubuloglomerular feedback (TGF) permits readjustment of the relationship of nephron filtration rate [single nephron glomerular filtration rate (SNGFR)] and early distal tubular flow rate (V(ED)) while maintaining TGF responsiveness. We used closed-loop assessment of TGF in hydropenia and after acute saline volume expansion (SE; 10% body wt over 1 h) to determine whether 1) temporal adaptation of TGF occurs, 2) adenosine A(1) receptors (A(1)R) mediate TGF responsiveness, and 3) inhibition of TGF affects SNGFR, V(ED), or urinary excretion under these conditions. SNGFR was evaluated in Fromter-Wistar rats by micropuncture in 1) early distal tubules (ambient flow at macula densa), 2) recollected from early distal tubules while 12 nl/min isotonic fluid was added to late proximal tubule (increased flow to macula densa), and 3) from proximal tubules of same nephrons (zero flow to macula densa). SE increased both ambient SNGFR and V(ED) compared with hydropenia, whereas TGF responsiveness (proximal-distal difference in SNGFR, distal SNGFR response to adding fluid to proximal tubule) was maintained, demonstrating TGF adaptation. A(1)R blockade completely inhibited TGF responsiveness during SE and made V(ED) more susceptible to perturbation in proximal tubular flow, but did not alter ambient SNGFR or V(ED). Greater urinary excretion of fluid and Na(+) with A(1)R blockade may reflect additional effects on the distal nephron in hydropenia and SE. In conclusion, A(1)R-independent mechanisms adjust SNGFR and V(ED) to higher values after SE, which facilitates fluid and Na(+) excretion. Concurrently, TGF adapts and stabilizes early distal delivery at the new setpoint in an A(1)R-dependent mechanism.     

The action of lipoxin-A on glomerular microcirculatory dynamics in the rat

Intrarenal administration of 750 ng/kg/min of LX-A in euvolemic rats resulted in significant increases in single nephron GFR (38.4 +/- 1.7 to 45.5 +/- 3.0 nl/min) and plasma flow rate (95 +/- 6 to 127 +/- 9 nl/min). The latter was due to a dramatic fall in afferent arteriolar resistance. Mean transcapillary hydraulic pressure difference increased from 33 +/- 1 to 43 +/- 3 mmHg (p less than 0.05) and the glomerular capillary ultrafiltration coefficient fell from 0.060 +/- 0.013 to 0.033 +/- 0.005 nl/(s X mmHg) (p less than 0.05). These responses to LXA in the renal microcirculation are in sharp contrast to those previously observed for the leukotrienes, and thus may represent the first example of counterregulatory (constrictor/dilator) vascular interactions within the lipoxygenase pathways.     

A rise in cyclic AMP with a lack of glycogenolysis by secretin in the isolated perfused rat liver and its inhibition by epinephrine

The effects of secretin on glucose output and cyclic AMP from the isolated perfused rat liver were investigated. Secretin 0.1 U/ml increased cyclic AMP in the effluent without an increase in glucose output. Glucose output induced by epinephrine 10(-8)M was not affected by secretin 0.1 U/ml administered simultaneously, whereas the increase in cyclic AMP produced by secretin 0.1 U/ml was inhibited by epinephrine 10(-8)M. The increase in cyclic AMP produced by glucagon 10(-10)M was not affected by epinephrine 10(-8)M. These results suggest that secretin does not affect glycogenolysis in the liver and secretin activates adenylate cyclase through a different receptor from glucagon in the liver.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Sweat lactate in exercising children and adolescents of varying physical maturity.

This study attempts to explain some of the individual variability in sweating pattern by comparing prepubescents and pubescents. Sweating rate and muscular anaerobic capacity are higher in adults than in children; thus we hypothesized that sweat gland anaerobic metabolism, as reflected by lactate excretion, might be higher with advanced physical maturity (PM). Lactate concentration in sweat ([LAC]sw) was measured at various stages of PM in boys who exercised in the heat. The subjects were divided into three groups on the basis of Tanner staging: prepubertal (PP, n = 16), midpubertal (MP, n = 15), and late pubertal (LP, n = 5). Subjects cycled at 50% of maximal O2 uptake for three 20-min bouts, with 10-min rest periods, in 42 degrees C and 18% relative humidity. Sweat samples were harvested, and population density of activated sweat glands was determined after each exercise bout. [LAC]sw during bout 1 was higher in PP than in LP [PP = 22.2 +/- 2.2, MP = 19.5 +/- 1.4, LP = 14.3 +/- 1.3 (SE) mmol/l]. In all groups, [LAC]sw decreased during subsequent bouts, and there were no intergroup differences in [LAC]sw during bout 3 (PP = 11.2 +/- 0.4, MP = 10.6 +/- 0.5, LP = 9.7 +/- 0.2 mmol/l). [LAC]sw was inversely related to sweating rate. Lactate excretion rate per gland was greater with the increase in PM (PP = 61.0 +/- 8.2, MP = 79.1 +/- 11.3, LP = 99.9 +/- 11.0 pmol/min; P = 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism.

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.     

Effect of the secretin family of peptides on gastric emptying and small intestinal transit in rats

We have compared the effects of the secretin family of peptides and their synthetic fragments on gastric emptying (GE) and small intestinal transit (SIT) using an unanesthetized rat model which simultaneously measures the GE and SIT of both solids and liquids. The meal consisting of 5% polyethylene glycol w/v, 5% Indian ink v/v and 20 non-digestible plastic beads was given intragastrically 10 minutes after the intraperitoneal injection of 0.5 ml of saline or peptides (2 and 5 micrograms/kg). Plasma secretin and the immunospecificity of secretin fragments were determined. In control rats, the t1/2 for the GE of both solids and liquids were 56 +/- 3.8 and 19 +/- 2.3 minutes, respectively. Liquids emptied faster than the solids and liquids travelled ahead of the solids in the intestine. Secretin (5 micrograms/kg) inhibited GE of both solids and liquids by 33-37%. Secretin delayed the SIT of the meal by approximately 35%. Fragments of secretin and of VIP had no effect on GE and SIT of both solids and liquids. The whole molecule of secretin was required to inhibit GE and to delay SIT of solids and liquids. Glucagon, PHI and growth hormone releasing factor (GHRF1-44) inhibited GE and SIT of both solids and liquids. For all peptides tested, the inhibition of SIT was proportional to the inhibition of GE suggesting that the prolongation of SIT was secondary to delayed GE. These observations indicate that the peptides of the secretin family inhibit GE and prolong SIT. Thus, the structural requirement required for the secretin family of peptides to effect their motor actions on the stomach is similar to that required for pancreatic enzyme secretion.     

Effects of secretin on insulin secretion and glucose tolerance.

Effects of intravenous (IV) infusion of secretin during IV infusion of glucose were examined in normal men. Secretin was administered according to three schedules: with each schedule a comparable priming dose was delivered in the first minute, but this was followed by a maintained (120 min) infusion of secretin at a relatively high rate, or by maintained infusion at one-third that rate, or by brief (15 min) infusion at the lower rate. The lower infusion rate produced increments in secretin in the blood within the range attainable during endogenous secretion. By comparison with effects of glucose alone each secretin infusion enhanced the increments of immunoreactive insulin in the blood. Enhancement of the early release (0-5 min) of insulin was similar with each type of secretin infusion, but the integrated changes in insulin levels through the total infusion period were related to the total doses of secretin. With each dose of secretin glucose tolerance was improved but the three mean glucose curves observed during infusions of secretin were not distinguishable from one another in spite of widely different integrated insulin responses. Secretin did not modify suppression of immunoreactive glucagon or free fatty acids in the blood during hyperglycemia. The results suggest that the effect of continuous administration of secretin on glucose tolerance is not simply related to its integrated insulinotropic action. It is suggested that the effect may be highly dependent on enhancement of insulin secretion early in the response to glycemia, or that it may be due to effects of secretin on glucose production or disposal which are not mediated by insulin.