Cyclic nucleotide-dependent phosphorylation of rat intestinal microvillus and basal-lateral membrane proteins by an endogenous protein kinase

0016-5085/78/75050838$02.00/0

Vol.75,

GASTROENTEROLOGY 75~838-846, 1978 Copyright 0 1978 by the American Gastroenterological Association

No. 5

Printed in U.S.A.

CYCLIC NUCLEOTIDE-DEPENDENT PHOSPHORYLATION OF RAT INTESTINAL MICROVILLUS AND BASAL-LATERAL MEMBRANE PROTEINS BY AN ENDOGENOUS PROTEIN KINASE LINDAJ. SHLATZ,DANIELV. KIMBERG,ANDKATHLEENA. CATTIEU Department

of Medicine,

University of Rochester

School of Medicine and Dentistry, Rochester,

New York

Both the microvillus and basal-lateral membrane components of intestinal epithelial cells were found to contain endogenous cyclic nucleotide-dependent protein kinases and their endogenous protein substrates. The phosphorylation of either membrane component using [Y-~‘PIATP as substrate, occurred very rapidly, reaching maximal levels at 1 min. Both cyclic AMP and cyclic GMP were shown to stimulate the phosphorylation of the microvillus and basal-lateral membranes; the approximate concentrations of cyclic AMP and cyclic GMP required for half-maximal stimulation of phosphorylation were 2 x lo-’ M and 1.7 x 1O-8M, respectively, for the basal-lateral membranes, and 2 x 1O-7 M. and 3.2 x lop8 M, respectively, for the microvillus membranes. Although both membrane components were phosphorylated by an endogenous protein kinase, the microvillus membrane was consistently phosphorylated to a greater extent at maximally effective concentrations of either cyclic nucleotide. The microvillus and basallateral membranes were also found to contain a phosphoprotein phosphatase; however, the rate of removal of [32Plphosphatefrom the microvillus membrane was found to be more rapid. Neither cyclic AMP nor cyclic GMP altered the activity of the enzyme in either membrane. The present results together with earlier studies are compatible with the possibility that the regulation of water and electrolyte transport in the small intestine by cyclic AMP and cyclic GMP may be mediated through modulation of the phosphorylation of protein components of the microvillus and basal-lateral membranes.

opposite to those produced by the cyclic AMP; active absorption of both sodium and chloride is enhanced and bicarbonate secretion is inhibited. 11,l2 Because these alterations in.ion transport could not be attributed to a decrease in the concentration of cyclic AMP in the mucosal cells, the existence of an additional regulatory system in the small intestine was suggested.13 Subsequent studies 14*I5demonstrated that muscarinic cholinergic and cz-adrenergic agonists increase the level of cyclic GMP in mucosal cells, and it has been suggested that the increase in this cyclic nucleotide may be associated with a stimulation in active absorption of sodium chloride. Cholecystokinin octapeptide and insulin were shown to cause similar elevations in cyclic GMP levels. l4 In view of the hypothesis that the biological effects of the cyclic nucleotides may be mediated through regulation of the activity of protein kinases,16 and the subsequent discoveries that cyclic nucleotide-dependent proReceivedMarch13, 1978. Accepted June14,1978. tein kinases were integral components of plasma memAddressrequestsforreprintsto: Dr.DanielV. Kimberg,Departbranes from a wide variety of tissues,“+’ it seems likely mentof Medicine,Collegeof Physicians andSurgeonsof Columbia that the phosphorylation and dephosphorylation of University, 630West168thStreet,NewYork,NewYork 10032. Thisstudywassupported by GrantAM-18912 fromtheNational unique membrane proteins could provide a mechanism for regulating membrane function and structure. InInstitute of Arthritis,Metabolism andDigestiveDiseases. Dr. Shlatz’spresentaddressis: MedicalCollege of Ohio, Toledo, deed, in 1972, Lucid and COX~demonstrated that cholOhio 43614. era enterotoxin enhanced the incorporation of phos-

The secretion of water and electrolytes in the small intestine is stimulated by several hormones, including secretin, glucagon, gastrin, vasoactive intestinal peptide, vasopressin, thyrocalcitonin, several prostaglandins, and the enterotoxins from Vibrio cholerue and Escherichia coli.’ Cyclic AMP’ has been suggested as a mediator for certain of these agents because this nucleotide has been shown to stimulate or unmask active intestinal secretion with inhibition of active sodium and chloride absorption and an increase in active chloride secretion and perhaps bicarbonate secretion. 2*3 A clearcut and direct relationship between the various secretory stimulants and cyclic AMP has been demonstrated only in the case of vasoactive intestinal peptide, prostaglandins, the enterotoxin of V. cholerue, and the heatlabile enterotoxin of E. coli. &lo In contrast, epinephrine and norepinephrine cause changes in active intestinal ion transport that are

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ENDOGENOUS INTESTINAL MUCOSAL PROTEIN KINASES

phate into the brush border fraction of rat small intestinal mucosal cells. More recently, de Jonge31described cyclic AMP and GMPdependent phosphorylation of a brush border protein from rat intestine with an apparent molecular weight of 86,000. Studies with purified basal-lateral membranes were not undertaken. 3L In view of the possible relationship between the level of membrane phosphorylation and the permeability and transport properties of the intestinal epithelial cell, the present study was undertaken to determine whether the purified plasma membranes of the enterocyte Ouminal or microvillus versus basal-lateral) possessed the necessary components of a phosphorylating system: an endogenous cyclic nucleotide-dependent protein kinase, endogenous protein substrates, and a phosphoprotein phosphatase. Microvillus and basal-lateral membranes were isolated and compared with respect to protein kinase and phosphoprotein phosphatase activity and cyclic nucleotide regulation of these enzymes. Materials and Methods Materials. All radioactive compounds and reagents for scintillation counting were obtained from New England Nuclear Corporation, Boston, Massachusetts. ATP, GTP, cyclic AMP, and cyclic GMP were supplied by Boehringer Mannheim Corporation, New York, New York. Sucrose, grade I; bovine serum albumin; N-2-hydroxyethyl-piperazine-N’-2ethanesulfonic acid (HEPES); imidazole; ouabain, phosphoenolpyruvate; pyruvate kinase, type III; and neutral alumina, WN-3, were purchased from Sigma Chemical Company, St. Louis, Missouri. Dowex AG 50 WX 4, 200-400 mesh, was obtained from Bio-Rad Laboratories, Richmond, California. Caffeine and triethanolamine-HCl were purchased from Eastman Organic Chemicals, Rochester, New York, and theophylline was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. Glucostat Special Reagent was supplied by Worthington Biochemical Corporation, Freehold, New Jersey. All other chemicals were of reagent grade. Preparation of intestinal epithelial cells. Nonfasting male Sprague-Dawley rats, weighing 200 to 275 g, were killed by cervical dislocation and the small intestine was immediately removed. Isolated intestinal cells were prepared from the proximal 40 cm according to the method of Stern3*as modified by Murer et a1.33;this method, which employs a buffered citrate solution, isolates only epithelial cells and excludes serosal and interstitial cells. After incubation with the citratecontaining solution, this buffer was discarded and the intestine was refilled with the appropriate buffer (see below). The cells were then dissociated in the appropriate buffer required for further subcellular fractionation. Preparation of basal-lateral membranes. Isolated enterocytes (approximately 20 g of wet weight) were washed three times by sedimentation at 500 x g for 10 min in 10 ml of icecold 0.25 M sucrose, 10 mM triethanolamine-HCl, 0.5 mM ethylenediaminetetraacetate (EDTA) buffer, pH 7.5 (STE), and then homogenized with 25 strokes of a tight-fitting Dounce homogenizer in 10 volumes of the same buffer. The homogenate was then diluted 1:l with cold STE and basallateral membranes were subsequently prepared by the method of Murer et a1.34with the modification that a tight-fitting Dounce homogenizer was utilized in all homogenization procedures. After discontinuous sucrose density gradient centrifugation, the basal-lateral membranes, which collect at the

839

30%/20% interface, were removed with a Pasteur pipet, diluted with additional STE buffer, and pelleted by centrifugation at 200,000 x g for 30 min. The final membrane fraction was resuspended in appropriate buffer for morphological and enzymatic analysis. Preparation of microvillus membranes. Enterocytes (4 g of wet weight) were dissociated in 10 to 20 ml of 5 mM EDTA, pH 7.4, and collected by centrifugation at 500 x g for 10 min. Isolated enterocytes were homogenized in 75 volumes of icecold 5 mM EDTA, pH 7.4, with 75 strokes of a tight-fitting Dounce homogenizer. The brush border fraction was obtained by the method of Forstner et a13’and the microvillus membranes were subsequently separated from the fibrous core components and the terminal web by a modification of the method of Hopfer et al.% Purified brush borders were suspended in 20 ml of 100 mM nmannitol, 1 mM Tris-HEPES buffer, pH 7.5, and reisolated by centrifugation at 24,000 x g for 20 min. The pellet was resuspended in 20 ml of the mannitol, Tris-HEPES buffer and homogenized with 25 strokes of a tight-fitting Dounce homogenizer. One milliliter of 2 mM MgSO? was immediately added and the homogenate was blended on a Vortex mixer. The homogenate was then centrifuged at 4,500 x g for 10 min. The resulting supernatant was then centrifuged at 24,000 x g for 30 min. The pellet containing the purified microvillus membranes was resuspended in appropriate buffer for morphological and enzymatic analysis. Assay ofprotein kinase activity. Protein kinase activity was determined by a modification of the procedure of Kuo.~ The standard assay mixture in a final volume of 0.1 ml contained: 50 mM sodium glycerol phosphate, pH 7.0; 10 mM MgCl,; 10 mM NaF; 2 mM theophylline; 5 PM [r-““PIATP (1.0 &i); either 2 cogof homogenate protein or 1 pg of membrane protein; and when indicated either 0.5 pM cyclic AMP or 0.05 PM cyclic GMP. The mixture was incubated for 1 min at 30” C unless otherwise indicated, and the reaction was terminated by the addition of 1.0 ml of ice-cold 5% trichloroacetic acid. After standing at 0”C for 10 min, the mixture was filtered through an 0.45 w Millipore (HA) filter. The filter was then washed twice with 10 ml of 5% trichloroacetic acid. After drying, the filter was placed in 10 ml of New England Nuclear Formula 950A scintillation solution and the radioactivity was measured in a Beckman LS-350 liquid scintillation spectrometer. All assays were performed in triplicate. Assays of marker enzyme activities. Na+,K’-ATPase activity, defined as the difference between total adenosine triphosphatase and Mgt2-dependent adenosine triphosphatase activities, was measured by a modification of the method of Siegel and Goodwin.= The incubation mixture contained 10 mM imidazole-HCl, pH 7.1, 10 mM MgCIZ, 5 mM ~Y-~~P]ATP(0.1 pCi), and 1 to 4 /~g protein with and without 120 mM NaCl and 20 mM KC1 in a final volume of 40 ~1. Reactions were carried out at 37”C for 15 min and terminated by the addition of 10 4 of cold 50% trichloroacetic acid. The released “P, was measured as described. I’ Adenylate cyclase activity was determined by a modification of the method of Salomon et al. 4(’ The incubation medium contained 50 mM Tris-HCl, pH 7.5, 5 mM MgCl,, 10 mM caffeine, 10 mM phosphoenolpyruvate, 12.5 g of pyruvate kinase, 1.2 mM [o-3ZP]ATP (0.4 &I), and 15 to 80 pg of protein in a final volume of 50 ~1. Samples were incubated at 37”C for 10 min. Isolation of the cyclic AMP was achieved by a combination of chromatography on Dowex 50 AG WX 4 cation exchange resin (0.4- x 6.0-cm column) and neutral alumina (0.4- x 2.5-cm column) as described by method C.“’ Guanylate cyclase activity was measured by a modification

840

SHLATZ

of the method of Krishna and Krishnan.41 The incubation medium in a final volume of 0.1 ml contained 40 mM Tris-HCl, pH 7.4, 3.3 mM MnC12, 1 mM cyclic GMP, 2 mM theophylline, 1 mM ~w~*P]GTP,and 15 to 60 pg of protein. Incubations were carried out at 37”C for 15 min. Dissacharidase activity was determined by a modification of the method of DahlqvisF using sucrose as the substrate. The glucose formed was measured with Glucostat Special Reagent. NADPH cytochrome c reductase was measured by the method of Masters et a1.43Succinate dehydrogenase was assayed spectrophotometrically by recording the amount of succinate oxidized in the presence of ferricyanide at 420 nm according to the method of King. 44 Protein concentration was determined by the method of Lowry et al. 4i using bovine serum albumin as the standard.

Results Characterization of microvillus and basal-lateral membranes. The microvillus membrane of the luminal

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ET AL.

tion was confirmed by electron microscopy which showed the preparation to consist only of large open sheets of trilaminar structure. Neither membrane preparation was significantly contaminated by nuclei, mitochondria, or endoplasmic reticulum as demonstrated by the lack of DNA, insignificant succinate dehydrogenase activity, or NADPH cytochrome c reductase activity, respectively. Distribution of protein kinase activity in the membrane components. As shown in table 2, both the mi-

crovillus and basal-lateral membranes contained an endogenous, cyclic nucleotide-dependent protein kinase and its endogenous protein substrates. Protein kinase activity, therefore, is not exclusively localized at one pole of the cell membrane, but is distributed throughout the cell membrane, as is Mg+*-ATPase.

Characterization of the [“*PIphosphate incorporated into membranes. Treatment of the [32Plphosphorylated

surface contains disaccharidases, alkaline phosphatase, membranes with 0.8 M hydroxylamine at pH 5.3 did not and leucyl napthylamide hydrolase,34,~5,46*47 whereas release the incorporated 13*Plphosphate,indicating that the basal-lateral membrane contains Na+,K+-ATPase the phosphate is not associated with membrane proteins and 5’-nucleotidase.3e 47-4L, In addition, adenylate cy- through labile acyl phosphate bonds, and suggesting clase and guanylate cyclase have been shown to be that the linkage to membrane proteins may be through localized largely in the basal-latera13”j” and microvil- phosphor-y1esters. This was further suggested by the ~us~‘-~~membranes, respectively, and in the intestine finding that phosphorylation of the membranes was not the guanylate cyclase activity is almost entirely mem- inhibited by the presence of 1 mM ouabain (results not brane-bound. shown). Time course of phosphorylation of microvillus and Table 1 depicts the enzymatic profiles of the isolated microvillus and basal-lateral membranes. The microv- basal-lateral membranes. Under the standard assay illus membranes employed in this study demonstrated conditions, the endogenous phosphorylation of both the a parallel enrichment in disaccharidase and guanylate microvillus and basal-lateral membranes reached a cyclase activities, 14.7-fold and X-fold, respectively, maximal level rapidly in the presence or absence of 0.5 when compared to the homogenate; they did not contain PM cyclic AMP or 0.05 PM cyclic GMP as shown in significant adenylate cyclase or Na+,K+-ATPase activi- figures 1 and 2. The basal rate of 13*Plphosphateincorties. The degree of purification of this membrane com- poration into the microvillus membranes was linear up ponent is comparable to microvillus membranes pre- to 2 min, whereas that of the basal-lateral membranes pared in previous studies utilizing mucosal scrapings,% was linear up to 5 min. Although the stimulator-yeffects although somewhat lower than that reported by Hopfer of cyclic AMP and cyclic GMP were observed at all et al.% Because our homogenate contained neither Tris incubation times tested, the percentage of stimulation nor HEPES, both of which inhibit disaccharidase activ- by the cyclic nucleotides was invariably greater at the ity, the initial activity is approximately 50% higher earlier time points. As a result of these data, a 1-min than that found in the presence of either of these period of incubation was chosen for the standard assay buffers, thereby reducing the apparent purification fac- procedure. Effect of membrane protein concentration on phostor when compared to other studies.% The endogenous phosphorylation of miIn comparison, the basal-lateral membranes ex- phorylation. hibited a 12.6-fold increase in Na+,K+-ATPase activity crovillus and basal-lateral membranes in the presence and a 14.7-fold increase in adenylate cyclase activity or absence of maximal concentrations of either cyclic with respect to the homogenate; these membranes did AMP or cyclic GMP was proportional to the amount of not contain significant disaccharidase activity. Lack of purified membrane protein below 1.0 pg (figs. 3 and 4). microvillus contamination in the basal-lateral prepara- It is important to note that the total amount of TABLE 1. Distribution Fraction Homogenate Microvillus membrane Basal-lateral membrane

of enzyme activities in membrane components

of intestinal epithelial cells”

Mg+*-ATPase (~mol/min/mg)

N+,K+-ATPase @nol/min/mg)

Adenylate cyclase (pmollminlmg)

Disaccharidase (wol/min/mg)

Guanylate cyclase (pmol/min/mg)

0.102 k 0.009 0.358 k 0.008 0.308 + 0.032

0.028 ? 0.003

6.11 ” 3.34 0.25 + 0.2 89.9 + 10.6

0.081 2 0.006 1.19 2 0.035 0.007

20.8 k 5.4 312.2 k 19.8

a Results expressed are the mean value k SE.

0.361 ? 0.06

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November 1978

INTESTINAL

TABLE2. Protein kinase distribution in membrane components of intestinal epithelial cells” Proteinkinasc Fraction _ cyclicnucleotide + cyclicnucleotide Homogenate Microvillus membrane Basal-lateral membrane

30.1 2 1.6 54.5 t 2.6

84.2 t 8.9 141.7 5 7.9

44.9 + 6.9

112.5 ? 9.9

(1Incubation conditions were as described in “Materials and Methods.” Protein kinase activity was determined in the presence and absence of 0.5 FM cyclic AMP or 0.05 PM cyclic GMP. Results are expressed in picomoles per minute per milligram and represent the mean value 2 SE

MUCOSAL

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assessed (results not shown). Neither cyclic nucleotide appeared to significantly alter the concentration of ATP required for half-maximal phosphorylation of either the microvillus or basal-lateral membrane. From these data, it was estimated that the concentration of ATP required for half-maximal phosphorylation of the microvillus membrane was 3.9 x 10” M in the presence and absence of 0.5 ~.LMcyclic AMP or 0.05 PM cyclic GMP. Similarly, values of 4.8 x lo* and 3.1 x 10” M were estimated as the concentration of ATP required for half-maximal phosphorylation of the basal-lateral membrane in the absence or presence of cyclic nucleotide, respectively. It should be emphasized that these ATP concentrations should not be equated with K, values because the presence in the membrane preparations of interfering enzymes, such as ATPases and protein phosphatases, complicates any quantitative interpretation of the data. Effect of varying concentrations of cyclic AMP and cyclic GMP on phosphorylation. The effect of varying

concentrations of cyclic AMP and cyclic GMP on the endogenous phosphorylation of microvillus and basallateral membranes is shown in figures 5 and 6. The concentration of cyclic AMP required for half-maximal phosphorylation of microvillus and basal-lateral mem---

I

2

T

1

1

6

0

---

4

6

8

10

TIME (min) FIG. 1. Time course of phosphorylation of microvillus membranes in the presence (0) and absence (01 of either 0.5 pM cyclic AMP or 0.05 PM cyclic GMP. Incubation conditions were as described under “Materials and Methods” except that the reaction times were varied as indicated. In this figure and in others in which 0.5 PM cyclic AMP or 0.05 WM cyclic GMP were used, the data represent a composite because at these concentrations the effects of the two nucleotides were identical.

1”‘Plphosphate incorporated in all of these experiments was quite small. Accurate measurement required that the substrate, lr-32PlATP, be of high specific activity. Effect of ATP concentration on phosphorylation. The effect of cyclic AMP or cyclic GMP on the endogenous phosphorylation of microvillus or basal-lateral membranes as a function of the concentration of ATP was

0 control ?? cyclic nucleotlde

0

IO

5

TIME

15

(mln)

FIG. 2. Time course of phosphorylation of basal-lateral membranes in the presence (01 and absence (0) of either 0.5 p,M cyclic AMP or 0.05 pM cyclic GMP. Incubation conditions were as described under “Materials and Methods” except that the reaction times were varied as indicated.

SHLATZ ET AL.

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was then diluted with buffer, reducing the ATP concentration and thereby minimizing any additional protein kinase activity during the ensuing incubation period. Both the microvillus and basal-lateral membranes exhibited phosphoprotein phosphatase activity as shown in figures 7 and 8. Phosphoprotein phosphatase activity appeared to be greater in the microvillus membrane because 50% of the incorporated 13*Plphosphatehad been removed in 3 min, whereas this degree of dephosphorylation required 7 min in the basal-lateral membrane preparations. Neither 0.5 PM cyclic AMP or 0.05 PM cyclic GMP altered the activity of the phosphoprotein phosphatase in either membrane fraction. Discussion Cyclic AMP and cyclic GMP have been suggested as mediators regulating the secretion of water and electrolytes in the small intestine, and are known to act by modulating the activity of protein kinases in other systems.21’ **,54-5sAn earlier report had suggested that cholera toxin-induced secretion in rat small intestine was correlated with an increase in the in vivo incorporation of [32P]phosphate into the brush border.30 Furthermore, a more recent study by de Jonge31 demon-

0

2

MEMBRANE

6

4

PROTEIN

(pg)

FIG. 3. Effect of the concentration of membrane protein on the [32P]phosphateincorporation into the microvillus membranes in the presence (0) and absence (01 of either 0.5 ELM cyclic AMP or 0.05 HAM cyclic GMP. Incubation conditions were as described under “Materials and Methods” except for the variation of the microvillus membrane protein concentration.

branes was estimated to be 2 x lo-’ M, and maximal activation was observed in the presence of approximately 5 x lo* M cyclic AMP in both instances. The concentrations of cyclic GMP required for half-maximal phosphorylation of microvillus and basal-lateral membranes were approximately 3.2 x lops M and 1.7 x 10m8 M, respectively; maximal stimulation of the microvillus membrane was observed at a cyclic GMP concentration of 5 x lo-’ M, whereas maximal stimulation of the basal-lateral membrane occurred at a concentration of 1 x lo-’ M. Although the apparent K, values for cyclic AMP and for cyclic GMP in the two membranes were similar, the cyclic nucleotides consistently stimulated the phosphorylation of the microvillus membrane to a greater extent. Incubation in the presence of both cyclic AMP and cyclic GMP, at concentrations providing maximal stimulation of protein kinase activity when added alone, did not show additive effects on the activity of the enzyme. Dephosphorylation of microvillus and basal-lateral membranes. In order to measure the rate of removal of

[32P]phosphate from the microvillus and basal-lateral membranes, membrane protein was preincubated for 1 min with [y-32PlATP under standard assay conditions to allow for maximal phosphorylation, and the sample

I

0

1

1

2

1

4

MEMBRANE

cyclic

I

nuclrotide

1

I

6

PROTEIN bg)

FIG. 4. Effect of the concentration of membrane protein on the [32Plphosphate incorporation into the basal-lateral membranes in the presence (0) and absence (0) of either 0.5 PM cyclic AMP or 0.05 PM cyclic GMP. Incubation conditions were as described under “Materials and Methods” except for the variation of the basal-lateral membrane protein concentration.

November

ENDOGENOUS

1978

INTESTINAL

A

A

---.

cycle GMP cydc AMP

MUCOSAL

PROTEIN

843

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either membrane fraction, and half-maximal stimulation of the microvillus or basal-lateral membrane was noted with 3.2 x lO+ M and 1.7 x lo+ M cyclic GMP, respectively. Although kinetic parameters and the apparent K, values for the two cyclic nucleotides were similar for the enzymes in both membranes, it would appear that the substrate protein content of the microvillus membrane may be greater because the level of phosphorylation in both the basal and cyclic nucleotidestimulated state was consistently higher. The possible participation of membrane phosphorylation in regulating membrane function and structure has been implicated in several systems, including synaptic transmission,“4r55 glucose transport in adipocytes,“, x and sodium transport in turkey erythrocytesS7*3HFurthermore, a role for membrane phosphorylation in regulating membrane permeability has been suggested in the toad bladde+ x and in kidney tubules. 29-2fi In renal proximal and collecting duct epithelial cells, the cyclic AMP-dependent protein kinase was localized primarily in the microvillus membrane,“4*2i and phosphorylation of the luminal surface was correlated with physiological data which suggested that this surface is modified during parathyroid hormone-induced changes in sodium, calcium, and phosphate transporttiH4 Of interest is the fact that the parathyroid hormone-sensitive aden-

zqk

0

10-a

10-7

10-6

CYCLIC NUCLEOTIDE CONCENTRATION (M) FIG. 5. Effect of increasing concentrations of cyclic AMP and cyclic GMP on the phosphorylation of basal-lateral membranes. Incubation conditions were as described under “Materials and Methods” except for the variation in the concentration of the indicated cyclic nucleotide.

r-l+’ I -

1

)

strated that low concentrations of cyclic GMP (0.05 to 1.0 PM) and somewhat higher concentrations of cyclic AMP (1.0 to 10.0 PM) stimulated the phosphorylation of a protein with an approximate molecular weight of 86,000 in isolated brush borders from rat intestine, but results with purified microvillus membranes free of core material were not presented. Of note, too, is the fact that de Jonge was unable to demonstrate cyclic GMPdependent phosphorylation of proteins in preparations presumably enriched in basal-lateral membranes, but these fractions were not characterized and the degree of enrichment is unknown. 31 In the present study, both highly purified microvillus and basal-lateral membranes were shown to possess endogenous protein kinases which could phosphorylate endogenous membrane proteins. Furthermore, the properties of the enzyme in either membrane component were very similar to each other and to those of many other cyclic nucleotide-dependent protein kinases in other tissues. 17,60,61The concentration of cyclic AMP providing half-maximal stimulation of either the microvillus or basal-lateral protein kinase was about 2 x lop7M. In comparison, cyclic GMP was considerably more effective in stimulating the phosphorylation of

1-

)-

1-

)-

1 -41

1

0

10-E CYCLIC

1

10-7 NUCLEOTIDE

10-6 CONCENTRATION

(M)

FIG. 6. Effect of increasing concentrations of cyclic AMP and cyclic GMP on the phosphorylation of basal-lateral membranes. Incubation conditions were as described under “Materials and Methods” except for the variation in the concentration of the indicated cyclic nucleotide.

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ET AL.

ylate cyclase in the renal tubule is localized to the basal-lateral membrane.65 It is possible that the intestinal epithelial cell response to hormonal regulation may also be polar in nature. Although the enzymes catalyzing the phosphorylation of the membranes were found to be present in both the microvillus and basallateral surfaces, this may be misleading for it does not take into account the possibility that the substrates in the two membranes may not be identical. In addition, it is also necessary to consider the accessibility of the membrane kinases to cyclic AMP or cyclic GMP, the concentrations of nucleotide available at each site, and the relative affinity and specificity of the kinases for each of the nucleotides. The enzymes catalyzing the formation of cyclic AMP and cyclic GMP are largely localized to the basal-lateral and microvillus membranes, respectively.34*5a-53and the relatively insensitive fluorescent immunocytochemical technique has provided evidence that, in the jejunum, cyclic AMP remains localized at the basal-lateral sides of the epithelial cells and in the lamina propria, whereas cyclic GMP

0 contrd ?? cychc nuclrotide

1

100

2

4

6

TIME

g

s

8o

1

60

g

40

20

0

2

10

12

(min)

FIG. 8. Effect of cyclic nucleotides on the rate of removal of [32Plphosphatefrom basal-lateral membranes. Incubation conditions were as described in figure 7.

Q P 4

8

4

8

__ IO

-J

TIME(min)6 FIG. 7. Effect of cyclic nucleotides on the rate of removal of 132P3phosphate from microvillus membranes. Membrane protein was preincubated for 1 min with [y-“PIATP under standard assay conditions to allow for maximal phosphorylation and then the sample was diluted to 1.0 ml with buffer. Samples were then incubated for the indicated times in the presence (0) and absence (0) of either 0.5 FM cyclic AMP or 0.05 FM cyclic GMP. Reactions were terminated by the addition of 1.0 ml of 50% trichloroacetic acid and the amount of phosphate remaining was determined as described in “Materials and Methods.”

remains in the area of the brush border membrane, smooth muscle, and nuclear elements.66The physiolog ical relevance of these observations in intact cells remains to be determined, however. The possibility that the level of membrane phosphorylation might be regulated by the cyclic nucleotides by altering the activity of a phosphoprotein phosphatase was also considered. Earlier studies with the toad bladder had demonstrated that cyclic AMP mediated the action of antidiuretic hormone in regulating sodium permeability by stimulating the dephosphorylation of a specific 50,000-mol wt protein in the membrane.21jXJIn the present study, both the microvillus and basal-lateral membranes were found to contain a phosphoprotein phosphatase; however, unlike the toad bladder membrane, the intestinal enzyme was not regulated by either cyclic AMP or cyclic GMP. Of note is the fact that de Jonge31also failed to find cyclic nucleotide regulation of the brush border phosphatase activity. The activity of the phosphoprotein phosphatase was greater in the microvillus membrane and this membrane was phosphorylated to a greater extent than the basal-lateral membrane. The rapid turnover of phosphorylated substrate in the microvillus membrane may indicate that phosphorylation at this cellular site might be more responsive to cyclic nucleotide regulation of transport processes. The relatively small quantities of phosphorylated protein, the rapidity with which these proteins were phosphorylated, the endogenous membrane location of

November

1978

ENDOGENOUS

INTESTINAL

these protein substrates as well as the enzymes involved in their phosphorylation, and the dependence of the specific phosphorylation reactions on the presence of cyclic AMP or cyclic GMP, all suggest that the process of phosphorylation of the membrane proteins may be related to important regulatory functions at the level of the cell membrane in the intact cell. Having characterised the protein kinases present in the small intestinal epithelial cell, the identification of the specific protein substrates in the microvillus and basal-lateral membranes remains to be elucidated. Furthermore, the functional significance of membrane phosphorylation in the hormonal regulation of intestinal secretion and absorption of water and electrolytes in intact cells must also be explored. REFERENCES Kimberg DV: Cyclic nucleotides and their role in gastrointestinal secretion. Gastroenterology 67:1023-1064, 1974 Field M: Intestinal secretion. Gastroenterology 66:1063-1084,

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16.

17.

1974 Sheerin HE, Field M: Ileal HCOs secretion: relationship to Na and Cl transport and effect of theophylline. Am J Physiol 228:1065-1074, 1975 Schaefer DE, Lust WD, Sircar B, et al: Elevated concentrations of adenosine 3’5’~cyclic monophosphate in intestinal mucosa after treatment with cholera toxin. Proc Nat1 Acad Sci USA 67:851-856, 1970 Kimberg DV, Field M, Johnson J, et al: Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J Clin Invest 50:1218-1230, 1971 Sharp GWG, Hynie S: Stimulation of intestinal adenyl cyclase by cholera toxin. Nature 229:266-269, 1971 Kimberg DV, Field M, Gershon E, et al: Effects of prostaglandins and cholera enterotoxin on intestinal mucosal cyclic AMP accumulation. J Clin Invest 53:941-949, 1974 Schwartz CJ, Kimberg DV, Sheerin HE, et al: Vasoactive intestinal peptide (VIP) stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54:536-544, 1974 Evans DJ Jr, Chen LC, Curlin GT, et al: Stimulation of adenyl cyclase by Escherichia coli enterotoxin. Nature [New Biol] 236:137-138, 1972 Guerrant RL, Ganguly U, Casper AGT, et al: Effect of Escherichia coli on fluid transport across canine small bowel. Mechanism and time-course with enterotoxin and whole bacterial cells. J Clin Invest 52:1707-1714, 1973 Dietz J, Field M: Ion transport in rabbit ileal mucosa. IV. Bicarbonate secretion. Am J Physiol 225:858-861, 1973 Field M, McCall I: Ion transport in rabbit ileal mucosa. III. Effects of catecholamines. Am J Physiol 225:852-857, 1973 Field M, Sheerin HE, Henderson A, et al: Catecholamine effects on cyclic AMP levels and ion secretion in rabbit ileal mucosa. Am J Physiol 229:86-92, 1975 Brasitus TA, Field M, Kimberg DV: Intestinal mucosal cyclic GMP: regulation and relation to ion transport. Am J Physiol 231:275-282, 1976 Tapper EJ, Powell DW, Morris SM: Effect of high dose carbachol on intestinal electrolyte transport (abstr). Gastroenterology 70:942, 1976 Kuo JF, Greengard P: An adenosine 3’,5’-monophosphate-decoli. J Biol Chem pendent protein kinase from Escherichia 244:3417-3419, 1969 Guthrow CE, Allen JE, Rasmussen H: Phosphorylation of an

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