Neuropeptide modification of chloride secretion in guinea pig distal colon

This study examined the effects of electrically stimulating submucosal neurons in the guinea pig isolated distal colonic mucosa and determined the effects of several peptides that are present in these neurons. Electrical field stimulation of muscle-stripped segments of guinea pig distal colonic mucosa, set up in Ussing flux chambers, evoked an increase in short-circuit current (Isc), of 371 +/- 31 MicroA.cm-2. The response to electrical stimulation was abolished by tetrodotoxin and significantly reduced by serosal furosemide. Atropine reduced, but did not abolish, the neurally evoked response. Addition of neuropeptide Y and galanin to the serosal bath had no effect on baseline Isc, but both evoked a concentration-dependent decrease in the neurally evoked secretory response. Vasoactive intestinal polypeptide evoked a concentration-dependent increase in basal (unstimulated) Isc that was reduced by furosemide and unaltered by tetrodotoxin. Neuropeptide Y, but not galanin, significantly reduced the secretory responses to vasoactive intestinal polypeptide and bethanechol. Somatostatin 201-995 and human calcitonin gene-related peptide had no effect on basal Isc nor did either alter the neurally evoked response. These results suggest that acetylcholine and non-cholinergic neurotransmitter(s) stimulate chloride secretion in the guinea pig distal colonic mucosa. This neurosecretory response may be modulated by neuropeptide Y and galanin that are found within submucosal neurons.     

The expression of gastric H+-K+-ATPase mRNA and protein in developing rat fundic gland

The proton pump H⁺-K⁺-ATPase is the final common pathway mediating the production and secretion of hydrochloric acid by gastric parietal cells. The present studies were undertaken to examine whether the expression of gastric H⁺-K⁺-ATPase mRNA and protein changes are associated with the development of H⁺-K⁺-ATPase activity in the rat fundic gland. H⁺-K⁺-ATPase activity was examined in rat fundic gland at different stages from gestational day 18.5 to postnatal 8 weeks. The expression of H⁺-K⁺-ATPase mRNA was detected by in situ hybridization using a digoxigenin-labelled RNA probe with a tyramide signal amplification system. The expression of H⁺-K⁺-ATPase protein was evaluated by immunoblotting and immunohistochemistry using antibodies against H⁺-K⁺-ATPase - and -subunits. We found that H⁺-K⁺-ATPase enzyme activity was detectable from the onset of gland formation (day 19.5 of gestation) and increased with age in the developing rat fundic gland. Expression of mRNA and protein was also discernible at the same time, and a progressive increase in expressions was observed as rats developed. Our results suggested that in developing rat fundic gland, the expression of both mRNA and protein of H⁺-K⁺-ATPase increased with age in a manner that parallels the development of H⁺-K⁺-ATPase enzymebreak activity.     

Na(+)-K(+)-ATPase alpha(2)-isoform expression in guinea pig hearts during transition from compensation to decompensation

Disturbance in ionic gradient across sarcolemma may lead to arrhythmias. Because Na(+)-K(+)-ATPase regulates intracellular Na(+) and K(+) concentrations, and therefore intracellular Ca(2+) concentration homeostasis, our aim was to determine whether changes in the Na(+)-K(+)-ATPase alpha-isoforms in guinea pigs during transition from compensated (CLVH) to decompensated left ventricular hypertrophy (DLVH) were concomitant with arrhythmias. After 12- and 20-mo aortic stenosis, CLVH and DLVH were characterized by increased mean arterial pressure (30% and 52.7%, respectively). DLVH differed from CLVH by significantly increased end-diastolic pressure (34%), decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (-75%), and increased Na(+)/Ca(2+) exchanger (25%) mRNA levels and by the occurrence of ventricular arrhythmias. The alpha-isoform (mRNA and protein levels) was significantly lower in DLVH (2.2 +/- 0.2- and 1. 4 +/- 0.15-fold, respectively, vs. control) than in CLVH (3.5 +/- 0. 4- and 2.2 +/- 0.13-fold, respectively) and was present in sarcolemma and T tubules. Changes in the levels of alpha(1)- and alpha(3)-isoform in CLVH and DLVH appear physiologically irrelevant. We suggest that the increased level of alpha(2)-isoform in CLVH may participate in compensation, whereas its relative decrease in DLVH may enhance decompensation and arrhythmias.     

Cardiopulmonary bypass reduces atrial Na+-K+-ATPase expression in children

Cardiopulmonary bypass (CPB) may induce serious side effects, potentially leading to myocardial failure. The Na(+)-K(+)-ATPase is a key component for myocardial function. Due to its developmental regulation, results from adult studies cannot be adopted to the situation in childhood. Right atrial myocardium from patients with left-to-right shunts at atrial level (VO, n=8) and those without (NO, n=8) was excised during heart surgery before and after CPB. Na(+)-K(+)-ATPase isoforms ATP1A1 (p=0.008) and ATP1A3 (p=0.038) decreased during CPB, which decrease was restricted to the VO group. This study highlights the importance of the underlying heart defect for susceptibility to the effects of CPB, showing a reduced Na(+)-K(+)-ATPase mRNA expression only in patients with left-to-right shunts on the atrial level. This seemed to be an early molecular event, as apart from one, none of the patients showed heart failure before or after surgery.     

Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl(-) secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na(+) absorption in distal colon. Amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive I(sc) and amiloride-sensitive (22)Na(+) absorption. The inhibitory effect of CCh was observed at approximately 0.1 microM, and maximum inhibition of approximately 70% was attained at approximately 30 microM (IC(50) = approximately 1 microM). CCh-induced inhibition of amiloride-sensitive I(sc) was almost totally abolished by 10 microM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive I(sc), but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca(2+) solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive I(sc), approximately 33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive I(sc), this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na(+) absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.     

Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

We investigated whether depressed muscle Na(+)-K(+)-ATPase activity with exercise reflected a loss of Na(+)-K(+)-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na(+)-K(+)-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at approximately 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na(+)-K(+)-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na(+)-K(+)-ATPase content via [(3)H]ouabain binding sites, and Na(+)-K(+)-ATPase alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, beta(2)- and beta(3)-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [(3)H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated alpha(1)-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Delta3-O-MFPase(rest-fatigue)) (r = -0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) alpha(1)-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Delta3-O-MFPase(rest-fatigue) (r = -0.56, P = 0.08). Exercise elevated alpha(2)-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Delta3-O-MFPase(rest-fatigue) (r = -0.60, P = 0.05). The average postexercise alpha(2)-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Delta3-O-MFPase(rest-fatigue) (r = -0.68, P < 0.05). Nonsignificant correlations were found between %Delta3-O-MFPase(rest-fatigue) and other isoforms. Thus acute exercise transiently decreased Na(+)-K(+)-ATPase activity, which was correlated with increased Na(+)-K(+)-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na(+)-K(+)-ATPase activity with exercise.     

Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus.

A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.     

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Studies on the glycoprotein component of (Na+-K+)ATPase from guinea pig brain

In this study an attempt was made to elucidate the possible mechanism of the brain microsomal (Na⁺-K⁺)ATPase inhibition based on the assumption that glycoprotein part of the enzyme is exposed on the outer membrane surface. In our experiments the modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in a significant decrease of the brain (Na⁺-K⁺)ATPase activity. The percentage of the enzyme inhibition by concanavalin A binding to the neuraminidase-treated preparation corresponds to the amount of liberated sialic acids. The modification of the glycoprotein part of the brain (Na⁺-K⁺)ATPase complex by neuraminidase and concanavalin A treatments did not affect K⁺-nitrophenylphosphatase activity.     

Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes

Functional expression of the rat colonicH⁺-K⁺-ATPasewas obtained by coexpressing its catalytic -subunit and the₁-subunit of theNa⁺-K⁺-ATPasein Xenopus laevis oocytes. We observedthat, in oocytes expressing the rat colonicH⁺-K⁺-ATPasebut not in control oocytes (expressing₁ alone),NH₄Cl induced a decrease in⁸⁶Rb uptake and the initial rateof intracellular acidification induced by extracellularNH₄Cl was enhanced, consistentwith NH⁺₄ influx via the colonicH⁺-K⁺-ATPase.In the absence of extracellularK⁺, only oocytes expressing thecolonicH⁺-K⁺-ATPasewere able to acidify an extracellular medium supplemented withNH₄Cl. In the absence ofextracellular K⁺ and in thepresence of extracellular NH⁺₄, intracellular Na⁺ activity in oocytes expressingthe colonicH⁺-K⁺-ATPasewas lower than that in control oocytes. A kinetic analysis of⁸⁶Rb uptake suggests thatNH⁺₄ acts as a competitive inhibitor of thepump. Taken together, these results are consistent withNH⁺₄ competition forK⁺ on the external site of thecolonicH⁺-K⁺-ATPaseand with NH⁺₄ transport mediated by this pump.

     

Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 microm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N(omega)-nitro-L-arginine (L-NA; 100 microM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.     

Mode of action of ANG II on ion transport in guinea pig distal colon

The effect of ANG II on mucosal ion transport and localization of ANG type 1 receptor (AT(1)R) in the guinea pig distal colon was investigated. Submucosal/mucosal segments were mounted in Ussing flux chambers, and short-circuit current (I(sc)) was measured as an index of ion transport. Serosal addition of ANG II produced a concentration-dependent (10(-9)-10(-5) M) increase in I(sc). The maximal response was observed at 10(-6) M; the increase in I(sc) was 164.4 +/- 11.8 microA/cm(2). The ANG II (10(-6) M)-evoked response was mainly due to Cl(-) secretion. Tetrodotoxin, atropine, the neurokinin type 1 receptor antagonist FK-888, and piroxicam significantly reduced the ANG II (10(-6) M)-evoked response to 28, 45, 58, and 16% of control, respectively. Pretreatment with prostaglandin E(2) (10(-5) M) resulted in a threefold increase in the ANG II-evoked response. The AT(1)R antagonist FR-130739 completely blocked ANG II (10(-6) M)-evoked responses, whereas the ANG type 2 receptor antagonist PD-123319 had no effect. Localization of AT(1)R was determined by immunohistochemistry. In the immunohistochemical study, AT(1)R-immunopositive cells were distributed clearly in enteric nerves and moderately in surface epithelial cells. These results suggest that ANG II-evoked electrogenic Cl(-) secretion may involve submucosal cholinergic and tachykinergic neurons and prostanoid synthesis pathways through AT(1)R on the submucosal plexus and surface epithelial cells in guinea pig distal colon.