Glutamine stimulates prostaglandin-sensitive Na+-H+ exchange in experimental porcine cryptosporidiosis

Glutamine Stimulates Prostaglandin-Sensitive Na+-H+ Exchange in Experimental Porcine Cryptosporidiosis ROBERT

A. ARGENZIO,*

J. MARC

RHOADS,?

MARTHA

ARMSTRONG,*

and

GUILLERMO

GOMEZ§

*Center for Gastrointestinal Biology and Disease and Departments of Anatomy, Physiological Sciences and Radiology, and “Animal Science, North Carolina State University, Raleigh: and ‘Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina

Background/Aims: Recent studies of piglet ctyptosporidiosis showed an injury-induced impairment of sodium-glucose cotransport and a prostaglandin-mediated inhibition of neutral NaCl absorption. Because glutamine has been shown to stimulate both neutral and electrogenic Na’ absorption, this study examined the mechanism of prostaglandin-mediated inhibition of NaCl absorption and the effect of glutamine on these processes. Methods: lleal mucosa from control and infected pigs was mounted in Ussing chambers for flux studies or incubated with [14C]glutamine or [14C]glucose for metabolism studies. Results: Glucose and glutamine induced equivalent increases, 2-2.5 pEq.crnm2. h-l, i n Na’ absorption and short-circuit current in control ileum. Despite a reduction in villous surface area to one third of the control, glutamine enhanced both neutral and electrogenic Na’ absorption in the infected ileum by 3.5 2 0.5 pEq.cmP2.hm1, whereas glucose was only half as effective (P < 0.05). In addition, glutamine was oxidized to CO2 at rates three times those of glucose. lndomethacin enhanced, whereas amiloride, prostaglandin E2, and Cl-free solutions inhibited the glutamine-induced neutral Na+ transport. Conclusions: Glutamine-stimulated neutral Na’ absorption is mediated by a prostaglandin-sensitive apical Naf-Hf exchange mechanism. The heightened Na’-H’ exchange and tissue oxidation of glutamine suggest that glutamine is superior to glucose for use in oral rehydration solutions.

C

ryptosporidium is an important

cause of diarrhea in both immunocompetent and immunosuppressed hosts.’ For example, a recent National Institutes of Health panel concluded that Cryptosporidium may now be one of the three most important enteropathogens causing diarrhea1 illness worldwide.* The diarrhea is usually moderate to severe and self-limiting, although recent studies associated Cryptosporidizlm with chronic diarrhea in immunocompetent children.3 In the immunosuppressed patient, the diarrhea is described as prolonged, choleralike, a significantly high incidence of and life threatening’,*; about 10% has been estimated in human patients with the acquired immunodeficiency syndrome.* Unfortu-

nately, there is no vaccine or antimicrobial presently effective; treatment rehydration.* Previous

studies

in our laboratory

the colostrum-deprived piglet

have shown

(agammaglobulinemic)

is an excellent

of this infection.‘36

therapy that is

involves oral or intravenous that

neonatal

model to study the pathophysiology The studies

showed

that a marked

loss of the villous surface area of the ileum was associated with impaired glucose-coupled Na+ absorption in cryptosporidiosis. However, a prostanoid-mediated inhibition of neutral NaCl absorption was also present. Indeed, infected tissues treated with the prostaglandin synthesis inhibitor indomethacin showed rates of neutral NaCl absorption

approximating

studies suggested anism

remained

Recent

studies

intact

tissue.

despite

severe villous

tine in some species, Na+ absorption

The mech-

atrophy.

by Rhoads et al.’ with piglet

showed that glutamine, neutral

rates of control

that the neutral NaCl absorptive

jejunum

the major fuel of the small intesstimulated in control

both

electrogenic

and

and rotavirus-infected

animals. Glutamine has also been shown to promote repair of injured intestine,8 possibly through its stimulation of proliferative events including enhanced NaC-H’ exchange and ornithine decarboxylase been shown in several cell lines.“’

activity,

which has

Prostanoids increase levels of cyclic adenosine 3’,5’monophosphate (CAMP), whose action. in intestine is to inhibit neutral NaCl absorption and induce electrogenic Cl- secretion.‘* In the renal epithelial cell line LC-PKlI PKE20, which expresses Na+-H+ exchange on both apical and basolateral membranes, CAMP leads to inhibition of both ion exchange activities.‘” Accordingly, the increased levels of prostanoids in cryptosporidiosis may inhibit the action of glutamine in both the stimulation of Na+ transport and epithelial repair. The present examined the mechanism of the eicosanoid-mediated

study in-

Abbreviations used in this paper: kc, short-circuit current; J,,, mucosa-tmserosa flux; J,,, serosa-twmucosa flux; Jf& net sodium flux; QO,, tissue oxidation. 0 1994 by the American Gastroenterological Association 0016-5085/94/$3.00

GLUTAMINE IN PORCINE CRYPTOSPORIDIOSIS

June 1994

hibition these

of Na+ transport

and the effect

of glutamine

on

Experimental

animals

Animal

The spontaneous

were newborn

thereby

removed

from

preventing

two isolation

the

(Raleigh,

sow immediately

system,

1124 of the

at hourly

total

intervals,

conditions.

termination

after

of cryptosporidia’*

liquid

thereby

and rotavirus.‘s

for rotavirus,

All pigs used

for cryptosporidia.

described.3

a time

of 5 X 10’ oocysts was

shown

were obtained

metabolism segments

studies,

3 and 4 days after

to be at the peak

Pigs were killed by intracardiac

tal, and sections of ileum beginning junction

and treated

on day 3 of life. Control and infected

previously

of

sodium pentobarbi-

equilibrate;

and histological

studies,

analysis.

substrate

Formalin-fixed

in paraffin, cut into 7-pm-thick

tions, and stained with H&E for examination

then

if this differed discarded

sec-

by light micros-

by >25%

isotopic

taken from the reservoirs.

(period

2). The second period

mucosal

E2 [PGE,])

period.

Time control

Samples

control

pigs were measured

mean diameter. dimensional

to determine

These measurements

parameters

using equations

surface area, as described

villi per millimeter the total

of villi in a square of control

Methods

used

In dualto 36C1

from the total. Unidi-

Na+ and Cl- fluxes from mucosa to serosa (Jm,) and (Jrm) were calculated

Conductance

was calculated

current

using

standard

from the spontaneous

(1s~) or by clamping

the tissue

the PD.

Substrate Oxidation Studies Measurements

of glucose

to yield an estimate

of

2 mL of HCO,--free

Ringer’s

of mucosal

the mucosal surface tissue.

for in vitro

treat-

scintillation

counter.

of ‘2Na counts

and subtracted

on l.l-cm’

in this laboratory

when possible,

and

to yield

studies

in detail.’ Briefly, a piece of ileum

the second flux stable fluxes dur-

scintillation

CO1 were performed

and infected

0.1 mmol/L

for 2LNa in a crystal

of

millimeter

of

in the same tissue.

at + 100 /.tA for 5 seconds and recording

to three-

Ussing Chamber Studies have been described

therefore,

The number

surface. These values were used to estimate area per square centimeter

height

for a cylinder

previously.5

was also obtained

number

villous

were converted

and

during

the contribution

counts was determined rectional

amiloride,

indicated

compared

experiments,

of addition

or 10m6 mol/L serosal prosta-

experiments

were counted

20-

flux period

to the serosal reservoir.

mucosal

and for 36CI in a liquid

PD and short-circuit

villi from eight infected

mannitol

ing the entire two flux periods;

counter

after 30

by a second treatment,

were also studied

were statistically

for were

to the mucosal bath, balanced

or serosal bumetanide,

glandin

20 minutes

and zero time samples

usually consisted

(1 mmol/L

paired

the tissues were

period, and second 30-minute

of 30 mmol/L

equations.”

Morphometric Analysis

a flux period,

30 mmol/L glucose or glutamine of drugs

bathing

to 36C1

to their conductance;

Samples were then removed

equilibration

for

period

**Na and

After allowing

l), followed

minute

Effects

a 30-minute

according

elec-

that corrected

or serosal solutions

standards

(flux period

clamp

Ag-AgCI

“‘Na or both

during

equilibration,

calomel elec-

through

were given

from the experiment.

in

(PD) was measured

to matched

voltage

the isotopes

from serosa to mucosa

Four to five well-oriented

difference

tissues. Tissues were matched

labeling

copy.

villous

Tissues

the solutions

at 39°C.

connected

an automatic

fluid resistance.

ments

10 cm above the ileocecal

for in vitro transport

were embedded

using

by addition

An inoculum

given orally to the piglets

trodes,

minutes

and control pigs were

pigs were taken in pairs for in vitro studies infection.’

diet by

establishing

Cryptosporidiumpm~urn’~ oocysts were obtained

inoculation,

birth,

Rectal swabs were taken daily for de-

in the study were negative

potential bridges

100% 0, was used. A

and circulate

maintained

were added to the mucosal

NC). Pig-

300 mL/kg body wt daily given as

steady-state

as previously

by the

and placed in one of

They were fed a synthetic

a computer-driven

always negative

Carolina State

at the College of Veterinary

access to colostrum,

units.

piglets

were approved

North Carolina State University

lets were

crossbred

North

NC). All procedures

Care and Use Committee

Medicine,

solutions,

trodes, and the PD was short-circuited

from the College of Agriculture, (Raleigh,

reservoirs

using Ringer-agar

Animals and Housing

University

in HCO,--free

water-jacketed

Materials and Methods

obtained

the solutions;

gas lift was used to oxygenate

processes.

1419

was

glucose

or glutamine

‘*C]glutamine, well-oxygenated

to in

containing

5 mmol/L

and 2 PCi of [U-‘*C]glucose

or [U-

respectively. Solutions and tissue samples were with 100% 0,; the flasks were then stoppered a hanging

paper

0.3N

incubated

and glutamine

solution

with caps containing saturated

oxidation

circles of tissue incubated

with

in a shaking

water

and 0.1 mL of 70% HCI04

center

BaOH,

well in which

was placed.

a filter

Flasks were

bath at 39°C for 60 minutes, was added

to stop the reaction.

stripped of its muscle layers and mounted in Ussing chambers. Tissues were bathed on both surfaces with 10 mL of a Ringer’s

The flasks were then incubated for another 60-minute period to allow the CO, to diffuse into the center wells. The center

solution containing (mmol/L): Na+, 142; K+, 5; Ca, 1.25; Mg, 1.1; Cl-, 124; HCO,-, 25; HP04, 1.65; and H,PO*, 0.3. Then 10 mmol/L serosal glucose was osmotically balanced with

well containing

10 mmol/L

mucosal

mannitol.

HCO+--buffered

solutions,

95% 02/5%

the trapped

counter.

‘*CO2 was then counted

The same procedure

using

in a liquid appropriate

Cl- was

standards and blanks (l*C compounds in the absence of tissue) was also performed simultaneously. Adjacent circles of tissue

(5 mmol/L) choline. In

were taken for dry matter determination, and the results were reported as micromoles of substrate oxidized per gram of dry

In some experiments,

replaced with isethionate, HC03with HEPES and isethionate (20 mmol/L), and Na+ with

scintillation

CO* was used to gas

tissue weight

per hour.

GASTROENTEROLOGY Vol. 106, No. 6

1420 ARGENZIO ET AL.

Table1. Morphometric Infected

Measurements

Piglet

of Control

of an increase

diminished

Control

Parameter Villous height (pm) Villous surface area (W? No. of villi/mmz Mucosal-serosal surface area ratio

result

and

Ileum

fected animals,

Infected

770 ? 70

accompanied

280 5 60”

tional 3.07 k 0.4 x lo5 54.8 2 5.5

1.0 k 0.3 x 105a 60.8 k 3.9

16.8 -c 2.8

in JmS; however,

in infected

tissue.

the increase

increase

genie Na’ absorption glucose

NOTE. Results are expressed as mean k SE; n = 8. Mucosal surface area is based on the villous surface area (not including the microvillous area) times the number of villi in a square millimeter. af < 0.01 vs. control.

cotransport

Analysis

Resutts

Morphometric

for control

and infected

pigs are shown

obtained

in Table

for control

1. Villous

height

and surface area were reduced to one third of the control by the infection. Taking into account the total number of villi

in a square

villous

amplification

millimeter

of mucosal

factor of the mucosal

surface,

the

surface area

was also reduced threefold by the infection. As shown in Figure 1, control ileum differs from infected tissue in that it contains

vacuolated

cells have been shown cytosis.” They disappear fection but are restored

epithelium.

These vacuolated

to have the capacity completely in recovered

for pino-

3-4 days after intissue by 9 days

after infection.> Present measurements indicate that these cells comprise the distal 63% ? 9% of the control villus and 0% + 0% of the infected villus.

Effect of Glucose and Glutamine and Cl- Fluxes

on Naf

Previous dose response studies with piglet jejunum had shown that 30 mmol/L of mucosal glucose or glutamine produced maximal increases in Nat transwould be port.‘” These relatively high concentrations relevant to those present in oral rehydration solutions. Therefore, this concentration was selected for study. Table 2 compares basal fluxes of Na+ and Cl-, Isc, and conductance before and after the addition of 30 mmol/L glucose to the mucosal bath in both control and infected animals. As shown previously,’ net Na+ and Cl- absorption was impaired and conductance was reduced in the Cryptosporiditlm-infected ileum. Glucose increased net Na+ absorption in both control and infected tissue as a

on Nat

tissue was very

consistent

mechanism

Glutamine

with a gluta-

established

increased

Cl- fluxes, a result differing

for hu-

both unidirectional

from glucose;

however,

the

fluxes increased by approximately the same magnitude so that the net flux was not significantly affected. to glutamine

measurements

cotransport

man intestine.”

In infected

Mucosal Morphology

of sodiuminfection.

similar to that elicited by 30 mmol/L glucose. Glutamine induced an increase in net Nat transport that was equivamine-sodium

Results ate reported as mean -C SE, and statistical comparisons were performed with Student’s t test for paired or unpaired observations, as appropriate.

impairment

As shown in Table 3, the effect of glutamine

lent to that in the Isc, a result

Statistical

electro-

for several species’” and

in the cryptosporidial

fluxes, Isc, and conductance

was

affected.

with glucose-coupled

described

was

and in-

in Isc; unidirec-

fluxes were not significantly

These results are consistent

confirm earlier results indicating

6.1 + 1.9”

control

in net Na+ absorption

by an equivalent

or net Cl-

this response

In both

duced

tissue, both Na+ and Cl- fluxes in response differed from control

significant

increases

absorption, and the (3.4 + 0.5 pEq.crn-‘.

fluxes. Glutamine

in both

increment

net

net Na’ Na+

in-

and Cll absorption

hh’) exceeded the change in Isc (1.3 ? 0.1 pEq.cm-‘. hh’). Therefore, in infected ileum, glutamine appears to stimulate electrogenic Nat absorption, as well as an electroneutral The glutamine-induced

NaCl absorptive

process.

increases in unidirectional

Cl-

fluxes could represent selective increases in paracellular anion conductance2’ or simultaneous increases in specific Cl-

absorptive

and

secretory

processes.

For example,

Cooke and Dawson23 have shown that alanine was capable of inducing an electrogenic Cl secretory process in newborn rabbit ileum; also, alanine appears to be an activator of Cl- channels in enterocytes.24 Accordingly, it is possible that a portion of the glutamine-induced increase in Jsm for Cl and change in Isc represents electrogenic Clsecretion. Alternatively, disulfonic stilbene-sensitive, neutral

Cl- transport

pathways

in both mucosa-to-serosa

and serosa-to-mucosa directions have been described in rabbit colon.“5 To test the possibility that glutamineinduced Jsm for CIl was electrogenic, mucosal glutamine was added to paired tissues of control ileum in the presence or absence of lo-” mol/L serosal bumetanide, an inhibitor of electrogenic Cl- secretion.26 However, the change in Isc in the presence of bumetanide (1.5 ? 0.5 PEq. cm-* .h-‘) did not differ from that obtained in its absence (1.2 -+ 0.4 PEq. cm-2. h-l; n = 4), suggesting that electrogenic Cl- secretion was not involved. To additionally test the above assumption and to determine the nature of Na+ and Cl coupling, we performed studies in which Naf or Cl- were selectively

GLUTAMINE

June 1994

IN PORCINE

CRYPTOSPORIDIOSIS

1421

Figure 1. Control and infected piglet ileum. (A) Distal tip of control ileum from a B-day-old piglet. Epithelial cells in the distal two thirds of the villus are vacuolated, comprising a surface area of 1.93 2 0.3 x lo5 pm’. (B) Infected ileum from a 7day-old piglet (4 days after inoculation). Epithelial cells are cuboidal and nonvacuolated. Organisms can be found as small, black dots on the surface of the epithelial cells. Lamina propria is increased in width, and an inflammatory cell infiltrate is present (H&E; original magnification x132). By 9 days after inoculation, vacuolated cells reappear in parallel with restoration of villous height and architecture.5

omitted

from

the

bathed in lo-”

bathing

shows net Naf transport Cl--free

solutions.

mol/L indomethacin

conditions

All

tissues

mine-stimulated

were

and change in Isc in normal and

in the presence

or absence

increment

in

net

Naf

absorption

matched the increment in Isc and was identical to the increment obtained in normal Ringer’s, In contrast, in-

(see below). Figure 2

fected tissue treated

of gluta-

with glutamine

displayed

a blunted

mine. In both control and infected tissue, W-free conditions reduced net Na+ absorption compared with that of tissues exposed to normal Ringer’s solution. In control

change in net Na+ flux in Cl -free solution compared with that of tissues bathed in normal Ringer’s (P < 0.05). Also, the increment in Na+ flux change was now equiva-

ileum bathed with solutions

lent to the change

rendered

Cl--free,

the gluta-

in Isc, as shown

for the control.

In

Table 2. Effect of Mucosal Glucose on NaCl Absorption in Control and Infected Pigs Cl- transport (pEq,

Na’ transport (@f~h~1-cm~2)

h-1~cm-2) G

Group

n

Control +glucose Infected +glucose

12 12 9 9

J*rr

JrnS 13.9 17.2 8.9 10.5

? 2 2 IT

2.1 l.7b l.ld 1.1”

11.8 12.4 8.9 9.1

2 2 ? 2

J*s

J“et 1.2 0.9 0.7 0.8

2.1 4.8 0.03 1.4

? ? ? *

1.1 1.5b 0.8” l.la

9.3 8.7 6.6 7.9

? -c + +

1.2 1.0 0.6d 0.7

J*IT 7.3 7.7 7.3 7.5

* t & *

0.7 0.5 0.3 0.4

J“et 2.0 1.0 -0.6 0.3

-e ‘2 5

Isc 0.6 0.5 0.5’ 0.9

1.7 4.2 2.0 3.5

* 2 + +

0.3 0.3’ 0.3 o.7b

(mS/c~) 22.6 26.6 18.1 18.7

-t 2 2 +

1.9 1.5b 1.3d 1.0

NOTE. Paired tissues were studied with normal Ringer’ solution during the first period; 30 mmol/L glucose was then added to the mucosal solution, and a second flux period was obtained. Ten-millimolar glucose was used to bathe the serosal surface throughout. Glucose was osmotically balanced with mannitol in the opposite solution. Results are shown as mean + SE; n = number of animals. G, conductance; J,,,, net flux. “P < 0.05, ‘? < 0.01; glucose vs. Ringer’s ‘P < 0.05, dP < 0.01; control vs. infected.

1422

GASTROENTEROLOGY Vol. 106, No. 6

ARGENZIO ET AL.

Table 3. Effect of Mucosal

Glutamine

on NaCl Absorption

in Control and Infected

Pigs

Cl- transport (@q. h-l, cmm2)

Na’ transport (p.Eq. h-l, cm-‘)

G n

Group Control +glutamine Infected +glutamine

JIn8

13.1 14.7 a.1 12.1

11 11 15 15

JSnl

+- 1.0 k 1.0 t o.ac t 0.96

11.3 10.9 a.7 9.3

+ + + t

Jnls

Jnet

0.9 0.6 0.6 0.4

1.8 2 0.7 3.8 5 0.88 -0.6 k 0.5” 2.8 2 0.86

a.5 12.3 5.9 10.9

2 + + t

J“et

JSrn

0.8 1.2b 0.4” l.lb

7.6 12.0 7.2 9.7

0.5 0.7b 0.5 0.9*

k t k 2

0.9 0.3 -1.3 1.3

t + + 2

ISC

0.7 1.0 0.6’ 1.1”

1.2 3.5 1.6 2.9

k +k 2

( mS/cm2)

22.1 5 1.0 25.0 z 1.1 15.0 + 1.2” 20.9 k 0.86

0.2 0.5b 0.1 0.3a

NOTE. Paired tissues were studied with normal Ringer’ solution during the first flux period; 30 mmol/L glutamine was then added to the mucosal solution, and a second flux period was obtained. Ten-millimolar glucose was used to bathe the serosal surface throughout. Glutamine or glucose was osmotically balanced with mannitol in the opposite solution. Results are shown as mean 2 SE: n = number of animals. aP < 0.05, bP < 0.01; glutamine vs. Ringer’s “P < 0.01; control vs. infected.

Naf-free

Ringer’s,

cantly different

net Cl-

absorption

from zero (P > 0.10)

absence of glutamine

(data not shown). These results indi-

cate that both Na+ and Cland glutamine

-

was not signifiin the presence or

stimulation

INDO-Ringer-

are necessary for the basal

AJ2

-

Cl-Free

-

we

process as

Effect of lndomethacin and Glutamine Our previous piglet

results

of Cryptosporidkn-infected

ileum’ had shown that elevated

levels of tissue

impaired the action of a neutral NaCl absorp-

tive mechanism. mine-induced

Therefore, increase

be antagonized

we reasoned that the gluta-

in neutral NaCl

by endogenous

ingly, all subsequent L indomethacin

absorption

prostaglandins.

may

Accord-

studies were performed with 1 pmol/

in the tissue baths. As shown previously,’

indomethacin

enhanced

tissue (P < 0.05)

net Na+ absorption

and diminished

in infected

the Isc in both control

and infected tissue (P < 0.01, compared with indometha-

t

Y w” s

Therefore

electroneutral

AIsc, in which JEc is net Na+ flux.

-

prostanoids

T

Isc can be fully ac-

Na+ absorption.

define the glutamine-stimulated

of neutral Naf or Cl- absorp-

4 Control

1

tion and that the glutamine-induced counted for by electrogenic

4

tin [Figure

Infected

31 and without

also shown in Figure absorption

[Table

stimulated

identical

of indomethacin

to those obtained

(see Table

in the absence

3); in addition,

between the changes in glutamine-induced was preserved. In contrast, the increment

1

by glutamine

0

significantly

the equality

AJ: and Isc

in Jz

in infected indomethacin-treated

stimulated tissue was

greater (P < 0.05) than in the infected tissue

studied without

-1

3}). As net Na+

and Isc in control tissue by amounts that were

quantitatively 2

indomethacin

3, glutamine

indomethacin

(compared

with results in

Table 3). This was caused by an increased neutral Jz -3

indomethacin

* JNs net

JNs net

Figure 2. Cl--free solutions inhibit basal and glutamine-stimulated neutral Na+ transport. Tissues from the same animal were bathed in indomethacin (INDO)-Ringer’s solution or in indomethacin-Ringer’s rendered Cl--free. Glutamine (m) (30 mmol/L) was added to mucosal solution during period 2. Note that glutamine-induced change in Na+ flux equaled that in kc in control tissues (m) either in indomethacinor Cl-free Ringer’s, whereas in infected tissue, glutamine-induced change in Na+ flux equaled change in kc only in Cl--free Ringer’s, Results are shown as mean + SE; n = 12. *P < 0.05, **P < 0.01 from indomethacin-Ringer’s.

in

(P < 0.05). As a result, total net Na+ absorp-

tion under these indomethacin-treated

conditions

was in-

creased in infected pig ileum to levels no different from control levels. As before, change in JEc in infected tissue greatly exceeded that of Isc, indicating of the stimulated

that the majority

Na+ transport was electroneutral.

Transport Inhibitor Studies Neutral methacin

NaCl

absorption

and glutamine

stimulated

could represent

port of NaCl or electrically

silent antiport

by indo-

coupled transsystems such

1424

GASTROENTEROLOGY Vol. 106, No. 6

ARGENZIO ET AL.

infected

animals,

respectively);

JS,,, was not significantly

affected. As also shown in Figure 4, PGE, inhibited Na+ absorption to the same degree as amiloride increased

the Isc in control

with indomethacin. gether

produced

Na+ absorption

and infected

Addition

treated

of PGEz and amiloride

no significant

additional

inhibition

toof

than PGE, alone. It should be noted that

similar changes in Nat absorption

were shown in control

tissue treated with PGE, or amiloride of indomethacin, tissue without

whereas

Na+

indomethacin

loride or PGE, Figure

tissue

Isc induced

net but

(reference

6 and unpublished

5 shows the changes

lation

of net Na+ absorption

fected tissue,

results

in net Na+ absorption

or

In agreement

with

postulated were

Control

to represent the previous

difficult

changes

to

studies,’

a PGE-induced

Cl-

and/or

interpret

HCO,-

further

because

(e.g., Table

the above was

secretion.’ Cl- fluxes

of simultaneous 3). We examined

by ion replacement

in PGE-stimulated

the

Isc that

results of unidirectional

in conductance

this hypothesis bumetanide

previous

4) showed

in in-

significantly but change in Isc.

Anion Secretion

However, 3~

and Isc. However,

PGE-Induced (Figure

stimu-

and PGEz diminished

Na+ absorption had no effect on the glutamine-induced

to ami-

data).

both amiloride

glutamine-stimulated

in infected

was not responsive

(in the flux period subsequent

nor PGE, had an effect on the glutamine-induced

but in the absence

movement

by glutamine

to that shown in Figure 4) in the presence or absence of amiloride or PGE,. In control tissue, neither amiloride

and serosal

tissue. As shown in Fig-

attenuated the ure 6, removal of either Cl- or HC03PGE-induced Isc. Bumetanide, at a dose (lo-” mol/L) that fully inhibits

the Na+-K+-Cl-

bit ileum,29 reduced similar

the change

to the reduction

tion of bumetanide effect. In contrast, free Ringer’s

cotransporter

in rab-

in Isc by 65%, a value

noted in Cl -free Ringer’s;

addi-

to Cl -free Ringer’s had no further addition of bumetanide to HCO,--

essentially

eliminated

the Isc response

to

PGE. Therefore, the Cl--dependent portion of the PGEstimulated Isc is likely mediated by a bumetanide-sensi-

Infected 100

20 0 Normal Ringer

Figure 5. Amiloride and PGE inhibit glutamineinduced increase in neutral Na+ transport in infected tissue. Results are shown as the change in net Na+ transport or kc induced by glutamine in indometha tin-Ringer’s (M) or in indomethacin-Ringer’s containing 1 mmol/L mucosal amiloride (IM) or 1 ymol/L serosal PGE? (a). Values are means + SE from tissues studied during period 2 and represent change in net Na+ transport or kc from values shown from period 1 in Figure 4. *P < 0.05 from indomethacin alone.

Ci Free

lic03Free

Figure 0. Bumetanide and Cl- or HC03- replacement inhibit the PGEinduced kc. Control tissues were bathed in normal Ringer’s containing 1 umol/L indomethacin or in indomethacin-Ringer’s in which Cl- was replaced with isethionate and HC03- replaced with HEPES and isethionate. One tissue of each pair was bathed with 0.1 mmol/ L bumetanide on the serosal surface. Then 1 pmol/L PGEZwas added to the serosal surface, and the change in Isc to a maximum was recorded. W, control; 10e4 mol/L serosal bumetanide. Results are means + SE from 8-10 pigs. **P < 0.01 from control (minus bumetanide); ‘P < 0.05 from normal Ringer’s; ttP i 0.01 from normal Ringer’s

GLUTAMINE IN PORCINE CRYPTOSPORIDIOSIS

June 1994

rive Na+-K+-Cl-

cotransport

mechanism

Control

on the serosal

membranes. The bumetanide-resistant and Cl--independent Isc may be a result of electrogenic HCO,- secretion. Recent studies of rabbit ileum by Minhas et a1.,29 in

14

which HCOs- secretion was determined that CAMP induced identical increases

10 -

tion and Isc in the presence

of either

12

directly, showed in HCOs- secreCl--free

64-

Glutamine and Glucose Oxidation Rhoads et a13’ suggested num was linked inhibitor

neutral

to glutamine

of glutamine

that the ability

NaCl absorption oxidation

metabolism,

of gluta-

in piglet inasmuch

jejuas an

amino-oxyacetate,

inhibited both the glutamine-induced tissue oxidation (QOJ and neutral NaCl absorption. However, the QO, induced by 5 mmol/L glutamine was no greater than that induced in control

by an equimolar tissue3’;

concentration

also, it was absent

1

8-

solutions

or serosal bumetanide.

mine to stimulate

1425

of glucose

in rotavirus-in-

c

; 0 L

Infected

i

14 -I 12108-

fected tissue unless glutamine and glucose were present together.31 In an attempt to clarify these issues, we exam-

6-

ined glutamine metabolism more directly by measuring ‘*CO2 production from li*C]glutamine and [i*Clglucose. As shown in Figure 7, 5 mmol/L glutamine was oxidized by excised ileum at rates at least twice those of 5 mmol/L glucose; however, the rates of glutamine oxidation were similar in control and infected tissue when expressed per milligram of dry tissue weight. Furthermore, the addition 5 mmol/L oxidation shown).

of both substrates

in concentrations

of

each yielded no further increase in glutamine compared with glutamine alone (data not

Glucose

Glutamine

Figure 7. Oxidation of glutamine is greater than that of glucose in both control and infected tissues. Tissues were incubated in hiCOB-free Ringer’s containing 5 mmol/L glutamine or glucose and the ap propriate l“C isotope. The 14C02 was trapped in a well containing BaOH, and its level compared with a standard amount of isotope and the appropriate blank (incubation with BaOH, in absence of tissue). Results are means + SE of 6 pigs. *P < 0.05 from glucose.

Effect of 5 mmol/L Glutamine on Na+ Transport The above studies suggested appreciable metabolism of glutamine at concentrations of 5 mmol/L, and studies of concentration dependence of glutamine on QO, in piglet jejunum indicated a maximal stimulation at this concentration. To test if this lower glutamine concentration also stimulated neutral Naf absorption, infected tissues were studied before and after addition of 5 mmol/L glutamine to both the mucosal and serosal baths. Glucose (10 mmol/L) was also present in the serosal solution. However, the basal net Naf absorption of -1.4 + 0.8 pEq.cm-* .h-’ was not increased further by glutamine (- 1.7 2 1 .O PEq. cm-*. hh’; n = 6), suggesting the absence of a close linkage between glutamine oxidation and Na+ absorption at this external glutamine concentration.

Mscusdon The present study shows that the severely impaired Na+ transport of the Cryptosporidium-infected pig-

let ileum can be restored and fully stimulated to levels identical to control ileum. Although glutamine promoted electrogenic Naf absorption in both control and infected tissues, this strong stimulation of Naf transport by glutamine in infected ileum appears to depend on neutral Na+ absorption and takes place despite the fact that the villous surface area for absorption is reduced to one third of the control (Table 1; References 5,6). These results are summarized in Figure 8 and indicate that glutamine is twice as effective as glucose in stimulating Naf transport per square centimeter of serosal surface area of infected tissue. As also shown in Figure 8, glutamine-induced neutral Na+ transport in the presence of indomethacin in infected tissue is three times the rate in control tissue if these values are expressed per unit of mucosal surface area. These figures emphasize the quantitative importance of the neutral mechanism in infected ileum. It is likely that neutral NaCl absorption in both the

1426

ARGENZIO

GASTROENTEROLOGY

ET AL.

Na TRANSPORT

A

-

-Control-

of glutamine infected

Infected -

in stimulating

tissue

indicate

treated

neutral

with

indomethacin

effect of inducing

No. 6

Na+ absorption

that increased prostaglandin

the additional

Vol. 106,

in

and further

concentrations

have

a bumetanide-sensitive

Cl- secretory process that does not appear to be inhibited by glutamine. The present

results

differ to some extent

with results

of Nath et al.‘” in rabbit ileum infected with an adherent, effacing strain of Escberichia co/i, RDEC-1. ies, both glutamine L stimulated

an electrogenic

and infected

ileum,

was much .c . m

$

1

1B

reduced

the net secretion infection

0.8

and glucose

absorptive

process in control

but the stimulation

of Na+ transport

in the infected

8

0.6

in the rabbit

transepithelial

study,

0

any detectable

from glutamine,

olism of glutamine B

Glc

Gln

I+Gln

B

Glc

Gln

I+Gln

Figure 8. Glucose- and glutamine-induced changes in electrogenic ) and neutral (W) Na’ transport per unit of (A) serosal or (6) mucosal surface areas in control and infected piglet ileum. Values for mucosal surface areas were based on the villous amplification factors calculated in Table 1 from measurements of villous surface area and the number of villi in a square millimeter of mucosal surface. B, basal; Glc, glucose; Gin, glutamine; I, indomethacin.

natal piglet HC03-,

or glu-

and rabbit metabo-

For example,

fluxes of glutamine

oxidation

production

of glutamine

of alanine

a minimal oxidized

or

metab-

tissue. The present

was extensively

ileum; as suggested

results by neo-

by Rhoads et al.,” mitowould generate

which could drive the apical transport

Two interesting

but as yet unexplained

H+ and events.

results

of the

present study were that despite the severe villous atrophy, apical Na+-H+ exchange induced by glutamine in infected

basal and glutamine-stimulated conditions is mediated and Cl--HC03exchange mechaby apical Naf-Hf nisms. Bumetanide, a potent inhibitor of NaCl cotrans-

rabbit.

suggesting

by rabbit

showed that glutamine chondrial

by the

with 14C or “N did not differ significantly,

glutamate

h

piglet

in glutamine

pig and adult

nor was there i

results between

lism in the neonatal determined

0.2

Furthermore,

induced

was not reversed with either glutamine

ileum may be caused by differences

0.4

tissue.

of both Na+ and Cl-

cose. These conflicting 8 Q r ii -

In their stud-

at a dose of 10 mmol/

tissue

was equal

to levels in the control

tissue.

Furthermore, glutamine appeared to stimulate both electrogenic and neutral Naf absorption in the former, whereas only electrogenic in the latter.

Nat absorption

was stimulated

port processes,26 was without effect on basal or glutamine-stimulated Na+ absorption, whereas 1 mmol/L

Relevant to the first of these results may be a difference in cell age on the villus of control and infected piglets.

amiloride, which at the dose used is specific for Na+-Hf exchange inhibition without affecting NaCl cotransport neutral Naf absorption in both systems, 32333inhibited

Moon et a1.35 showed with C3H‘)thymidine

week old piglet ileum that the mean age of the cell at vacuolation is 91 hours after DNA synthesis, whereas

basal and glutamine-stimulated tissue. The ionic interdependence of neutral Naf or Cl- absorption (Figure 2) is also consistent with these processes and suggests indirect coupling via the intracellular PH.~’ Further studies with piglet ileal membrane vesicles will be necessary to formally identify these processes; however, the present data are consistent with this model. Amiloride-sensitive, neutral Na+ absorption also appears to be the mechanism inhibited by PGEa because significant glutamine stimulation of neutral Na+ transport was eliminated by exogenous prostaglandins and/or amiloride. These results explain the greater effectiveness

the turnover rate of the villous epithelium is 9 days. These vacuolated cells disappear with age together with a faster turnover rate of the epithelium. As the result of a probable increase in epithelial turnover rate of damaged and recovering tissue,36 the cell type on the infected villus may represent a transitional stage between the undifferentiated crypt cell and the vacuolated, pinocytotic absorptive cell. We speculate that neutral Na+ absorption is primarily expressed in this relatively young, nonvacuolated cell. Such speculation is supported by earlier findings of DeJesus and Smith,37 who showed that the ratio of electrogenic to neutral Na+ transport dimin-

labeling

of l-

June 1994

ished

GLUTAMINE IN PORCINE CRYPTOSPORIDIOSIS

in parallel

with

the disappearance

of vacuolated

nation

of glutamine

use in oral rehydration

1427

solutions

in

epithelium on the ileum as the piglet got older. They also showed a much reduced efficiency of Na+ absorption

human patients with cryptosporidiosis is warranted, and we are presently conducting such studies with infected

in cells undergoing

piglets.

pinocytosis.

surface area of nonvacuolated the same in our control

Inasmuch epithelium

attenuates

this action

present results cryptosporidiosis

suggest that therapeutic may be more effective

tissue shown

minishing

Nagy showed

8A could be explained.

underlying

exchange et a1.,38 using that glutamine

the selective

oxidation

probably infected

or crypt comprise animals,

a greater

to CO, was increased junction

proportion

a COz-driven

pH could promote cells3” Although

stimulation

increased the similar

in

in intracellular

exchange

rates of glutamine

in these

tissue

dry weight

may be misleading

when

the control

villus

to 2 on the infected

villus,5

2.

3.

4.

whereas

the ratio of glutamine metabolism in isolated enterocytes to that in immune cells is approximately 3:I (600 vs.

5.

200 nmol . mg-’ . h- ’ 40-43). Thus, it can be calculated, surface area and weight measurements

based on present

and previous morphometric data,5 that glutamine oxidation per enterocyte is approximately two times higher in

6.

infected tissue if metabolic rates of lamina proprial cells remain constant. However, it also appears that higher

7.

concentrations of glutamine are necessary to induce increased Naf-H+ exchange than to induce high rates of glutamine

oxidation.

lated cells are needed glutamine metabolism

Thus,

additional

studies

8.

with iso-

to determine the significance of to intracellular pH and Na+-H+

9.

exchange. Glucose-based oral rehydration solutions have been one of the most important therapeutic advances in this century for the treatment of diarrhea1 disease. In the present enteric infection of the neonatal piglet, the value of glucose seems questionable because glucose-sodium cotransport was diminished in proportion to the loss of surface area. Accordingly, the finding that the neutral Na+ absorptive mechanism is intact and can be fully stimulated by glutamine is of practical importance. Although our present results in neonatal piglets can not be extrapolated directly to humans, adult human intestine possesses the capacity for both glutamine metabolism and Nat-H+ exchange.44*45 Thus, it seems that an exami-

The former

should

then

the

approaches to if aimed at di-

of epithelial

solutions

CAMP

containing

allow maximal

of both net Na+ transport

1. Fayer R, Ungar BLP. Cryptosporidium

oxidation

comparing

the action

of glutamine Therefore,

gluglutaand

References

in our control and infected tissues appear inconsistent with this proposal, glutamine oxidation rates based on control and infected tissue. For example, the ratio of enterocytes to lamina proprial cells varies from 10 on

Na+ absorption.

to oral rehydration

mine-induced stimulation epithelial repair.

cells

of the villus

decrease Naf-H+

tamine.

as opposed

cells. Because such transitional

or inhibiting

in addition

in infected tissue is also unclear. isolated cells from rat intestine,

75% in cells from the crypt-villous to villous

X lo5

study also shows that increased pros-

pm2, as calculated from Table 1 and Figure l), the similar rates of neutral Na+ transport in glutamine-stimulated

of Na+-H+

(-1

synthesis

neutral

The mechanism

piglets

The present

taglandin

in stimulating

in Figure

and infected

as the villous was essentially

IO.

Il.

12.

13.

14.

spp and cryptosporidosis. Microbial Rev 1986; 50:458-483. Laughon BE, Allaudeen HS, Becker JM, Current WL, Feinberg J, Frenkel JK, Hafner R, Hughes WT. Laughlin CA, Meyers JD, Schrager LK, Young LS. Summary of the workshop on future directions in discovery and development of therapeutic agents for opportunistic infections associated with AIDS. J Infect Dis 1991; 164:244-251. Phillips AD, Thomas AG, Walker-Smith JA. Ctyptosporidium, chronic diarrhea and the proximal small intestinal mucosa. Gut 1992;33:1057-1061. Meisel JL, Perer DR, Melign C, Rubin C. Overwhelming watery diarrhea associated with a cryptosporidium in an immunosup pressed patient. Gastroenterology 1976;70:1156-1160. Argenzio RA, Liacos JA, Levy ML, Meuten DJ, Lecce JG, Powell DW. Villous atrophy, crypt hyperplasia, cellular infiltration, and impaired glucose-Na absorption in enteric cryptosporidiosis of pigs. Gastroenterology 1990;98:1129-1140. Argenzio RA, Lecce J, Powell DW. Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis. Gastroenterology 1993; 104:440-447. Rhoads JM, Keku EO, Quinn J, Woosely J, Lecce JG. L-Glutamine stimulatesjejunal sodium and chloride absorption in pig rotavirus enteritis. Gastroenterology 1991; 100:683-691. Fox AD, Kripke SA, DePaula J, Berman JM, Settle RG, Rombeau JL. Effect of a glutamine-supplemented enteral diet on methothexateinduced enterocolitis. JPEN 1988; 12:325-331. Chen KY, Canellakis ES. Enzyme regulations in neuroblastoma cells in a salts/glucose medium: induction of ornithine decarboxylase by asparagine and glutamine. Proc Natl Acad Sci USA 1977; 74:3791-3795. McCormack SA, Tague, LL, Cragoe EJ, Johnson LR. Regulation of ornithine decarboxylase activity in LoVo cells. Am J Physiol 1990;258:G934-G941. Rhoads JM, Chu PT, Chen W, Berschneider HM, Paradiso AM. Lasparagine (ASN) and L-glutamine (GLN) stimulate Na/H exchange in lPECJ2 cells (abstr). Gastroenterology 1992; 102: A237. Powell DW. lmmunophysiology of intestinal electrolyte transport. In: Field M, Frizzell RA, eds. Handbook of physiology. Intestinal absorption and secretion. Volume IV. Bethesda, MD: American Physiological Society, 1991:591-641. Casavola V, Helmle-Kolb C, Muren H. Separate regulatory control of apical and basolateral Na/H exchange in renal epithelial cells. Biochem Biophys Res Commun 1989; 165:833-837. Payne P, Lancaster LA, Heinzman M, McCutchan JA. Identification of ctyptosporidium in patients with acquired immunodeficiency syndrome. N Engl J Med 1983;309:613-614.

1428

15.

ARGENZIO

ET AL.

GASTROENTEROLOGY

Lecce JG, Balsbaugh RK, Clare DA, King MW. Rotavirus followed by hemolytic enterpathogenic Escherichia co/i in weanling diarrhea of pigs. J Clin Microbial 1982;16:715-721.

31.

Upton SJ, Current WL. The species Cryptosporidium (Apicomplexa: Cryptosporidiiae) infecting mammals. J Parasitol 1985; 71:625-629. 17. Schultz SG, Zalusky R. Ion transport in isolated rabbit ileum. I. Short-circuit current and Na fluxes. J Gen Physiol 1964;47:567584.

32.

16.

18.

33.

34.

Kraehenbuhl JP, Campiche MA. Early stages of intestinal absorption of specific antibodies in the newborn: an ultrastructural, cytochemical, and immunological study in the pig, rat, and rabbit. J Cell Biol 1969;42:345-352.

35.

19. Rhoads JM, KeKu EO, Bennett LE, Quinn J, Lecce JG. Development of L-glutamine-stimulated electroneutral sodium absorp tion in piglet jejunum. Am J Physiol 1990; 259:G99-G107.

36.

20. Powell DW. Intestinal water and electrolyte transport. In: Johnson LR. ed. Physiology of the gastrointestinal Tract. New York: Raven, 1987:1267-1305. 21. Said HM, Voorhis KV, Ghisham FK, Abumurad N, Nylander W, Redha R. Transport characteristics of glutamine in human intestinal brush-border membrane vesicles. Am J Physiol 1989;256: G240-6245. 22.

Powell DW. Intestinal conductance and permselectivity changes with theophylline and choleragen. Am J Physiol1974;227:14361443.

37. 38.

39. 40.

23. Cooke HJ, Dawson DC. Transport characteristics of isolated newborn rabbit ileum. Am J Physiol 1978:234:E257-E261. 24. Giraldez F, Sepulveda FV. Changes in apparent chloride perme ability of Necturus enterocytes during the sodium-coupled transport of alanine. Biochem Biophys Acta 1987;898:248-252.

41.

25.

Smith PL, Sullivan SK, McCabe RD. Concentrationdependent effects of disulfonic stilbenes on colonic chloride transport. Am J Physiol 1986;250:G44-G49.

42.

26. O’Grady SM, Palfrey HC, Field M. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues. Am J Physiol 1987; 253:C177-C192.

43.

Donowitz M, Welsh MJ. Regulation of mammalian small intestinal electrolyte secretion. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven, 1987:1351-1388. 28. Binder HJ, Sandle GI. Electrolyte absorption and secretion in the mammalian colon. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven, 1987:1389-1418.

44.

27.

29.

Minhas BS, Sullivan SK, Field M. Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence, and effects of cyclic nucleotides. Gastroenterology 1993;105:1617-1629. 30. Rhoads JM, KeKu EO, Woodard JP, Bangdiwala SI, Lecce JG, Gatzy JT. L-Glutamine with &glucose stimulate oxidative metabolism and NaCl absorption in piglet jejunum. Am J Physiol 1992; 26:G960-G966.

45.

Vol. 106,

No. 6

Rhoads JM, KeKu EO, Lecce JG. Glutamine stimulates oxidative metabolism and Na absorption in neonatal pig jejunum (abstr). Gastroenterology 1990; 98:A553. Benos DJ. Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physiol 1982;42:C131-C145. Palfrey HC, Alper SL, Greengard P. Protein phosphotylation and the regulation of cation co-transport. J Exp Biol 1980;89:103115. Nath SK. Dechelotte P, Darmann D, Gotteland M, Rangier M, Desjeux JF. [15Nl_and [%]glutamine fluxes across rabbit ileum in experimental bacterial diarrhea. Am J Physiol 1992; 262:G312G318. Moon HW, Kohler EM, Whipp SC. Vacuolation: a function of cell age in porcine ileal absorptive cells. Lab Invest 1973;28:2328. Thake DC, Moon HW. Lambert G. Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs. Vet Pathol 1973; 10: 330-341. De Jesus CH, Smith MW. Sodium transport by the small intestine of new-born and suckling pigs. J Physiol 1974;243:211-244. Nagy LE, Pittler A, Kretchman N. Development of glutaminase along the villuscrypt axis in the jejunum of rat. J Pediatr Gastroenterol Nutr 1988: 7:907-913. Grinstein S, Rothstein A. Mechanisms of regulation of the Na/ H exchanger. J Membr Biol 1986;90:1-12. Ardawi MSM, Majzoub MG, Newsholine EA. Effect of glucorticoid treatment on glucose and glutamine metabolism by the small intestine of the rat. Clin Sci 1988; 75:93-100. Watford M, Lund P, Krebs HA. Isolation and metabolic charaderistics of rat and chicken enterocytes. Biochem J 1979; 178:589596. Newsholme EE, Newsholme P, Curi R, Challoner E, Ardawi MSM. A role for muscle in the immune system and its importance in surgery, trauma, sepsis and burns. Nutrition 1988;4:261-268. Ardawi MSM, Newsholme EA. Glutamine metabolism in lymphocytes of the rat. Biochem J 1983; 212:835-842. Marliss EB, Aoki ll, Pozetsky T, Most AS, Cahill GF. Muscle and splancnic glutamine and glutamine metabolism in postabsorptive and starved man. J Clin Invest 1971;50:814-817. Turnberg LA, Bieberdotf, FA, Morawski SG, Fordtran JS. Interrelationships of chloride, bicarbonate, and sodium and hydrogen transport in the human ileum. J Clin Invest 1970;49:557-569.

Received April 5, 1993. Accepted January 3, 1994. Address requests for reprints to: Robert A. Argenzio, Ph.D., 4700 Hillsborough Street, Raleigh, North Carolina 27606. Fax: (919) 8294465. Supported by grants ROI-DK 40584, KO&HD00945, and P30 DK34987 from the National Institutes of Health and NRICGP 91372046411 from the U.S. Department of Agriculture.