Heterogeneous isoactin gene expression in the adult rat gastrointestinal tract

Heterogeneous isoactin gene expression in the adult rat gastrointestinal tract

Heterogeneous lsoactin Gene Expression in the Adult Rat Gastrointestinal Tract REBECCA Department A. LIDDELL, MARK SYMS, and KIRK M. MCHUGH of A...

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Heterogeneous lsoactin Gene Expression in the Adult Rat Gastrointestinal Tract REBECCA Department

A. LIDDELL,

MARK

SYMS,

and

KIRK M. MCHUGH

of Anatomy, Thomas Jefferson University, Philadelphia, Pennsylvania

BacMround: Normal gastrointestinal development is a complex process involving the precise integration of multiple cell types. To gain a better understanding of these processes, the present study examined isoactin gene expression in the adult rat gastrointestinal tract. Methods: Northern blot analysis was performed on specified segments of the adult rat esophagus, stomach, small intestine, cecum, colon, rectum, and anus using actin isoform-specific complementary DNAs for all six vertebrate isoactins. Results: Smooth muscle and cytoplasmic isoactins were heterogeneously coexpressed in a segment-specific manner throughout the gastrointestinal tract. In addition, striated muscle isoactin expression was also detected in segments of the adult rat esophagus, stomach, colon, cecum, rectum, and anus. Histological analysis indicated that the adult rat esophagus, stomach, and anus contained significant quantities of skeletal muscle, providing a source for the striated muscle isoactins detected in these gut segments. A similar source of striated muscle isoactin expression in the cecum, colon, and rectum was not identified. Both coordinate and independent regulation of isoactin gene expression was observed in the gastrointestinal tract, although distinct patterns of autoregulation were absent. Conc/usions: This study represents the first complete analysis of isoactin gene expression in the adult rat gastrointestinal tract and provides the basis for future studies designed to investigate the factors responsible for these processes.

from

six independent

tinct

temporal

ment

and in the adult.

pression

N

make

embryonic germ layers as well as a precise series of spatial reorderings including embryonic flexion and

rotation of the midgut. Recent studies in our laboratory have begun to analyze the molecular mechanisms responsible for some of these complex processes by examining isoactin gene expression in the developing and mature gastrointestinal tract of the rat and mouse. Actin is expressed as six distinct isoforms in higher vertebrates that include the P-cytoplasmic, y-cytoplasmic, ‘y-smooth muscle, a-smooth muscle, u-skeletal, and a-cardiac. These six actin isoforms are derived

the

spatial

patterns

These

various

actin

for the differentiation of tissue and cell types.

Prior

studieslM3 have

isoactins

are ubiquitously

throughout

shown

of the muscle

tive

to be limited

tends

phenotypes.

The smooth

ily coexpressed testinal

system.

They

muscle

isoactin

the

during

that

the cytoplasmic

of a

in all cell types

isoactins

In con-

is more restric-

to specific

muscle

isoactins

muscle

However,

include

maturation

cell

are primar-

cells of the gastroin-

tract, vascular

do exist for the expression tins.

of ex-

excellent

and

muscle

in smooth

tract, urogenital

spiratory

isoforms

and into adulthood.

trast, expression and

develop-

patterns

coexpressed

development

in dis-

during

distinct

variety

several

system,

and re-

additional

of the smooth expression

caveats

muscle

isoac-

of the cl-smooth

the early stages of striated

mus-

cle myogenesis

as well as in a variety

of myofibroblas-

tic cell type?

and the expression

of the y-smooth

muscle

isoactin

Coexpression shown heart

in developing

of the striated

to be limited and skeletal

Studies

muscle

family showed

the stomach

isoactins

has been

and

expression

in the rat and mouse. that the cytoplasmic and mature

these studies

that the striated

sperm.” mature

have characterized

were the primary

in the developing

mature

muscle.‘0-‘2

and tissue-specific

multigene

addition,

and

to the developing

in our laboratory’

velopmental

cle isoactins gastrointestinal development requires the of multiple cell types from all three interaction

and

markers

analysis ormal

genes that are expressed

the de-

of the actin Northern

blot

and smooth

mus-

actin isoforms gastrointestinal

expressed tract. In

also made the novel observation

muscle

and intestine

isoactins

were coexpressed

of the developing

in

and ma-

ture rat. In situ analysis (K. M. McHugh, manuscript in preparation) indicated that expression of the smooth muscle isoactins was limited to the smooth muscle cells of the gastrointestinal tract, whereas additional studies’-3 suggested that the cytoplasmic isoacAbbreviations used in this paper: tcRNA, total cellular RNA. 0 1993 by the American Gastroenteroiogical Association 0016-5085/93/$3.00

348

LIDDELL

ET AL.

GASTROENTEROLOGY

tins were coexpressed

in all cell types found

developing

and mature

cise nature

and location

the striated

muscle

tract remains These

and

isoactin

studies

and

study

striated

colon,

cellular

RNA

segments,

rectum,

(1) to further of the actin

rat gastrointestinal and

muscle

of adult rat esophagus,

cecum,

The pur-

expression

rat gastrointestinal

tract

location

isoactin tract.

of the

expression

Specified

stomach,

seg-

small intestine,

and anus were harvested.

was isolated

Northern

from

blotted,

complementary

results

showed

distinct

specific

patterns

with

(cDNA)

organ-specific

of isoactin

Total

each of these bowel

and probed

isoactin-specific

all six

probes. and

through-

out the adult rat gastrointestinal

tract. In addition,

precise

source

expression

muscle

isoactins

was further information adult

in the adult

the

of striated

rat gastrointestinal

tract

characterized. This study provides regarding isoactin gene expression

novel in the

rat gastrointestinal

complexity

The

segment-

gene expression

of the “ectopic”

of

gene expres-

adulthood.

the extent

reported

in the adult

patterns development.

was twofold:

in the adult

(2) to determine

ments

into

the heterogeneous family

previously

of

that the rat

of isoactin

also maintained

characterize

shown distinct

throughout

patterns

pose of the present multigene

displayed

expression

organ-specific

sion were

expression

in the rat gastrointestinal

had clearly

intestine

gene

These

the

tract. The pre-

of the “ectopic”

isoactins

No. 2

to be clarified.

earlier

stomach

gastrointestinal

within

Vol. 105,

tract

previously

and suggests

a level of

unreported.

Materials and Methods Animals Both

male

(Zivic

Miller,

Adult

rats were

intestine,

cecum,

esophagus, cally ments

and

and the esophagus, rectum,

further in Figure

for RNA

rats tissues.

stomach,

and anus were excised.

small intestine,

and

as outlined

Sprague-Dawley

colon,

stomach,

measured

adult

PA) were used to obtain

killed,

segments were pooled ately frozen in liquid needed

female

Zielienople,

small The

and colon were specifi-

subdivided

into

1. Specific

organs

distinct

seg-

and organ

from approximately 14 rats, immedinitrogen, and stored at -70°C until

isolation.

cDNA

probes

specific

characterized

1.

Schematic

segments

was specifically measured ment

and published.

have

correspond

2 (~2),

stomach

for all six actin isoforms

representation

jejunum

of the specific gastrointestinal

isolated from the adult rat. Each gastrointestinal

segments

DNA Probe Construction been previously

Figure

to esophagus

esophagus

segment (J),

and excised. segment

2 (s2),

stomach

ileum (I), cecum

COlOn (TC), descending

(cE),

segment

The adult rat gastrointestinal

segment 3 (~3),

1 (EI),

stomach

segment ascending

3 (s3),

esophagus segment

seg1 (sI),

duodenum

(D),

colon (AC), transverse

COlOn (DC), rectum (R), and anus (A).

These include

a 125-base pair (bp) rat y-smooth muscle isoactin-specific,” a 187-bp rat a-smooth muscle isoactin-specific,” a 404-bp human P-cytoplasmic isoactin-specific,‘3 a 365-bp

tin-specific14 probe. All six of these probes are derived from the 3’ untranslated regions of their respective isoactin cDNAs and are of similar length and G/C content. Isoac-

human y-cytoplasmic isoactin-specific,‘3 a 136-bp human a-skeletal isoactin-specific,‘4 and a 171-bp &cardiac isoac-

tin-specific mid DNA

cDNA insert fragments were isolated from plasusing the appropriate restriction enzymes. Insert

August

ISOACTIN

1993

by standard agarose gel electrophoresis and electroelution using an Elutrap apparatus (Schleicher & Schuell, Keene, NH). A total of 50 ng of each insert was labeled by random priming to a specific activity of at least 1 X lo8 cpm * pg-’ * mL-’ before use in Northern blot analysis. DNA

was purified

RNA Isolation and Northern Blot Analysis

were stripped

mmol/‘L

Tris-HCl,

by successive

washes

in 95°C

150

pH 7.5.

examined.

Results and efprobes.

of segments

stomach,

duodenum,

that each isoactin-specific RNA (mRNA) weight.

molecular

staining

tcRNA

of the

were loaded

not shown). malized

Northern

after

hybridization quantities

respectively

blots were quantitated discussed.’

comparisons

be made

bromide

and methylene

that equivalent

and transferred,

as previously

quantitative could

membrane

used to verify

hybrid-

species of the

Ethidium

of the RNA gel before blotting

were both

of isoactin

of (data

and nor-

In this manner,

for all six actin

ce-

these studies probe

ized to a single messenger

rat

ileum,

and anus confirmed

appropriate

349

the adult

jejunum,

and showed

blue

GI TRACT

from

rectum,

semi-

gene expression

isoforms

examined

in

this study. Northern smooth

blot analysis

muscle

trointestinal tively.

isoactin

of y-smooth expression

tract is shown

Quantitative

expression

analysis

(Figure

muscle

muscle

isoactin

and the transverse

colon

of smooth

coexpressed tract.

muscle

segment

relatively

low levels

isoactin

1 and

bowel,

and rectum. observed

muscle

isoactin;

however,

nately

reduced.

regulated

This muscle

throughout

This

in the

pattern

is

for the y-smooth

the overall

of the smooth

peaks in

the ascending

of expression

to that

is greatly

1

and rectum.

identical

expression

segment

small bowel,

almost pression

of the

low levels of

rat stomach small

Expression

relatively

of the a-smooth

with

throughout

peaks in stomach with

in the esophagus,

Expression

2A and El, respec-

2C) shows that the smooth

rat gastrointestinal

the adult

in the adult rat gasisoactin

the adult

expression

and a-

muscle

are heterogeneously

y-smooth

muscle

in Figure

isoactins

esophagus,

All blots were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) using intensifying screens. Each Northern blot series was quantitated using an LKB Ultrascan XL Enhanced Laser Densitometer and the Gelscan XL program (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Multiple exposures were chosen for each Northern blot series to ensure that each lane was scanned in the established linear range for these studies. Standard Gelscan XL program parameters were established for the initial series of quantitation and maintained for the remainder of the blots analyzed. The quantitation values reported for each lane generally represent an average of at least two different scannings of the same blot to eliminate a variety of blot and/or band inconsistencies. The various quantitation values obtained for each of the four isoactins examined were normalized to a specific activity of 2.8 X 10” cpm . I.tg-’ 0 mL-’ and an exposure time of 96.5 hours. In this way, a relative comparison of the expression of all six actin isoforms could be made for the various bowel segments

ficacy

esophagus,

IN RAT

cum, colon,

colon

Densitometry

Prior studies have shown the specificity of the various isoactin-specific cDNA

blot analysis

staining

Total cellular RNA was isolated from 1.2 g of each specific gastrointestinal segment according to the methods of Chirgwin et a1.i5 For each sample, specific segments were pooled from multiple animals to minimize any animal-specific differences in expression. Total cellular RNA was quantitated spectrophotometrically, and these values were visually confirmed by agarose-formaldehyde gel electrophoresis in the presence of 100 pg/rnL ethidium bromide. In addition, equivalent transfer of total cellular RNA (tcRNA) was verified by methylene blue staining16 of the Northern blot after hybridization. Northern blot analysis was performed with 40 /.tg of total cellular RNA by the method of Thomas” using 2% agarose-formaldehyde gels and Biotrans nylon membranes (ICN Pharmaceuticals Inc, East Hills, NY). Two complete Northern blot series were run on a single gel and blotted to a single membrane to eliminate gel and blotting discrepancies between each Northern blot. Prehybridization, hybridization, and wash conditions were identical to those previously discussed” and were the same for each of the various probes used. When necessary, Northern blots

Northern

EXPRESSION

magnitude

result

of ex-

suggests

isoactins

that

is coordi-

the adult rat gastrointesti-

nal tract. Northern

blot

analysis

3A) and y-cytoplasmic indicates neously

that

of ecytoplasmic

(Figure

the cytoplasmic

coexpressed

tract. Quantitative

3B) isoactin isoactins

in the adult analysis

shows that P_cytoplasmic stomach

segment

1, jejunum,

pression

of the y-cytoplasmic

(Figure isoactin

(Figure expression

are heteroge-

rat gastrointestinal 3C) of these results expression

and ascending isoactin

peaks in colon.

Ex-

peaks in stom-

ach segment 2 and the ascending colon. This pattern is similar to that observed for the P-cytoplasmic isoactin, although the overall magnitude is significantly less and the peak in P-cytoplasmic isoactin expression observed in the adult rat jejunum is not as apparent for the y-cytoplasmic isoactin. In general, the pattern of cytoplasmic isoactin expression appears similar to that observed for the smooth muscle isoactins. These re-

350

LIDDELL ET AL.

GASTROENTEROLOGY Vol. 105, No. 2

A

El

E2

E3 Sl

S2

S3 DU JE

El

E2

E3 Sl

S2

S3 DU JE IL

El

EZ

W

S,

S2

s3

Cu

JE

IL

CE

AC

TC

IL

a,

R

CE AC TC DC RE AN

CE AC TC DC RE AN

AN

SAMPLES

sults suggest that expression tins

is coordinately

well as the smooth Northern 4A)

and

of the cytoplasmic

regulated muscle

blot analysis the a-cardiac

between

isoac-

themselves

as

isoactins. with (Figure

the a-skeletal 4B) isoactins

expression only

shows

that expression of the striated muscle isoactins in the adult rat gastrointestinal tract is primarily limited to segments of the esophagus and colon. Quantitative analysis (Figure 4C) of striated muscle isoactin expression in the adult rat gastrointestinal tract shows that expression of the a-skeletal isoactin peaks in esophagus segment 2 and the cecum and ascending colon of the large bowel. Expression of the cr.-skeletal isoactin decreases dramatically in stomach segments 1 and 2 to reach undetectable levels in stomach segment 3 and the small bowel. Moderate levels of cl-skeletal isoactin

reappear

to decrease

verse colon, (Figure

Figure 2. Northern blot analysis of y-smooth muscle isoactin (A) and a-smooth muscle isoactin (5) expression using 40 ug of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (E 1-E3), stomach l-3 (s lS3), duodenum (Du), jejunum (JE), ileum (IL), cecum, ascending colon (AC), transverse colon (TC), descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the y-smooth muscle isoactin (0) and a-smooth muscle isoactin (A) isoactins in the adult rat gastrointestinal tract. Quantitation was performed and normalized as discussed in Materials and Methods.

in the cecum

to relatively

descending

levels of a-skeletal

colon,

isoactin

and ascending

low levels

and rectum.

expression

colon

in the transModerate

are also seen in

the anus. Similar patterns of expression are observed for the a-cardiac isoactin with slight peaks in expression occurring in the esophagus and large bowel. These peaks in a-cardiac isoactin expression are much less distinct than those observed for the a-skeletal isoactin, and the overall magnitude of a-cardiac isoactin expression is significantly less than that observed for the a-skeletal isoactin. These results indicate that the striated muscle isoactins are coexpressed within the various bowel segments examined in this study and suggest that these actin isoforms are coordinately regulated in the adult rat gastrointestinal tract. The two

ISOACTIN

August 1993

EXPRESSION

IN RAT

GI TRACT

351

A

El

E2

E3 Sl

S2

S3 DU JE IL

CE AC TC DC RE AN

El

E2

E3 Sl

S2

S3 DU JE IL

CE AC TC DC RE AN

c Figure 3. Northern blot analysis of &cytoplasmic isoactin (A) and y-cytoplasmic isoactin (6) expression using 40 pg of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (El-E3),stomach l-3(sl-s3),duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending colon (AC), transverse colon (TC). descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the j3-cytoplasmic isoactin (0) and y-cytoplasmic isoactin (A) in the adult rat gastrointestinal tract. The y-cytoplasmic isoactin expression is multiplied by IO. Quantitation was performed and normalized as discussed in Materials and Methods.

peaks in striated tinct

from

cytoplasmic cle isoactins

muscle

isoactin

those observed isoactins,

ulatory

; i

e 5 40 g

20

O S*MPLES

appear

dis-

basis. This observation

muscle

and

more distinct

patterns

that the striated

mus-

ent at the tissue-specific

regulated

these

bowel

the importance

in the regulation

18-23 An analysis

H SC

expression

are independently

mechanisms

expression.

*(

for the smooth

suggesting

other actin isoforms. Prior studies have shown

‘O’

from

of autoreg-

of isoactin

of the y-smooth

gene

muscle/

segments

does not preclude of autoregulation

the fact that may be appar-

level for each of the various

examined.

Discussion Normal the integration

gastrointestinal of numerous

development embryonic

requires

processes

in-

a-smooth muscle ratio, the P-cytoplasmic/y-cytoplasmic ratio, the smooth muscle/cytoplasmic ratio,

volving a variety of developmental paradigms. In an effort to begin to understand these complex processes,

the cl-skeletal/a-cardiac ratio, and the total mRNA content of the various bowel segments

we examined isoactin gene expression in sequential segments of the adult rat gastrointestinal tract. Northern blot analysis of isoactin gene expression in the adult rat gastrointestinal tract shows distinct patterns of smooth muscle, cytoplasmic, and striated muscle isoactin expression. The y-smooth muscle isoactin is

isoactin did not

reveal any distinct patterns (Table 1). The wide degree of variation observed for these ratios suggests that the adult rat gastrointestinal tract independently regulates isoactin gene expression on a segment by segment

352

LIDDELL

ET AL.

El

El

GASTROENTEROLOGY

rat gastrointestinal

No. 2

E2 E3 Sl S2 S3 DU JE IL CE AC TC DC RE AN

E2

E3 Sl

S2

S3 DU JE

IL

CE AC TC DC RE AN

Figure 4. Northern blot analysis of o-skeletal isoactin (A) and a-cardiac isoactin (3) expression using40 pg of total cellular RNA isolated from adult rat gastrointestinal segments including esophagus l-3 (E 1 -E3), stomach l-3 (S 1 -S3), duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending COlOn (AC), VZVISverse colon (TC), descending colon (DC), rectum (RE), and anus (AN). The position of the 18s and 28s ribosomal standards were marked on the original blots. (C) Relative expression of the a-skeletal isoactin (0) and a-cardiac isoactin (A) in the adult rat gastrointestinal tract. Both the a-skeletal isoactin and a-cardiac isoactin expression are multiplied by 10. Quantitation was performed and normalized as discussed in Materials and Methods.

SAMPLES

the predominant

Vol. 105,

actin isoform expressed in the adult tract. The exceptions

to this obser-

striated muscle isoactins in the adult rat gastrointestinal tract such that each pair of these actin isoforms is

vation are the first two segments of the esophagus and

coexpressed

the jejunum where the a-skeletal

consistent

and P-cytoplasmic

in a similar

manner.

These

results are

with prior studies,‘*24*25 which have shown

isoactins predominate, respectively. By comparison, moderate to low levels of a-smooth muscle and P-cy-

that the various actin isoforms are coordinately coexpressed as distinct pairs in most tissues. The present

toplasmic isoactin expression and low to undetectable levels of a-skeletal, y-cytoplasmic, and a-cardiac isoactin expression are also detected in the adult rat

study also shows that coordinate patterns of regulation are observed between the smooth muscle and cytoplasmic isoactins, suggesting that common regulatory pathways may exist for these four actin isoforms. These observations provide additional evidence to support our prior hypothesis that the cytoplasmic isoactins play a significant role in smooth muscle differentiation, maturation, and function.’ In contrast, expression of the striated muscle isoactins appears dis-

gastrointestinal tract. These observations are consistent with our prior developmental studies and show the complex nature of isoactin gene expression in the adult rat gastrointestinal tract. Coordinate patterns of isoactin gene expression are observed for the smooth muscle, cytoplasmic, and

ISOACTIN

August 1993

Table 1. lsoactin mRNA Ratios in Bowel Segments Adult Rat Gastrointestinal Tract

1:l 2:1 9:1 13:l 17:l 21:1 7:1 0.5: 1 13:l 1l:l 4:1 9:1 7:1

48: 1 45: 1 45: 1 15:l

20: 1 25: 1 5:1 11:l

19 25 68 694 408 199 13 32 125 147 441 433 69

6:1 13:l

13:1

31 165

5:l 7:1 3:l

18:l 15:l 23: 1 32: 1 1l:l 28: 1 1O:l 43: 1 17:l 38:1 44:1 38:1 25: 1

2:1 3:1

33: 1 60: 1

a-SM

El E2 E3 Sl s2 s3 DU JE IL CE AC TC DC

0.5: 1 0.5: I 3:1 5:1 1l:l 1O:l 2:1 1:l 20: 1 8:1

RE AN

Total actin

SM/CYT

Y-W

gastrointestinal

a-SKI/ a-CAR

P-CYT/ y-CYT

Samples

of the

-

NOTE. Relative y-smooth muscle (y-SM), a-smooth muscle (a-SM), P-cytoplasmic (P-CYT), y-cytoplasmic (y-CYT). a-skeletal (a-SKL), and a-cardiac (a-CAR) isoactin expression in esophagus segment 1 (El), esophagus segment 2 (E2), esophagus segment 3 (E3), stomach segment 1 (Sl), stomach segment 2 (S2), stomach segment 3 (S3), duodenum (DU), jejunum (JE), ileum (IL), cecum (CE), ascending colon (AC), transverse colon (TC). descending colon (DC), rectum (RE), and anus (AN) of the adult Sprague-Dawley rat. Relative isoactin gene expression was quantitated and normalized as discussed in Materials and Methods. Ratios were rounded to the nearest whole number. CYT equates the sum of the normalized P-cytoplasmic and y-cytoplasmic isoactin expression. SM equates the sum of the normalized y-smooth muscle and a-smooth muscle isoactin expression. Total actin equates the sum of the normalized expression of all six actin isoforms in each bowel segment.

EXPRESSION

tract.

various

caveats

smooth

muscle

This

that

exist

isoactins

terns of smooth

of the

various

Similar

patterns

pression been

stripped

isoactins

observed

The

to cytoplasmic to establish

with

supported McHugh

these

isoactins,

either

the smooth

indicating

muscle

that independent

ways exist for these actin isoforms. autoregulation observed

in the adult

importance the

for the various

mented.‘8-23 most

by recent

significant

has

been

The of

Schevzov

functional cultured

these mechanisms

in whole

organ

it is also possible

that autoregulation

segments.

However,

does not play a

the expression

of the

sphincteric

(phasic)

The P-cytoplasmic adult rat gastrointestinal

in assessing

in isoactin smooth

gastrointestinal

asso-

hypothesis

is

(K. M. that have

gene expres(tonic)

and

muscle

tis-

tract. In ad-

myoblasts.

path-

rat gastrointestinal

This

et a1.23 recently reported distinct for the cytoplasmic isoactins in

autoregulatory difficulty

differences

functions

differences

heterogeneously

in the adult

in modulating

tract.

dition,

Such

in our laboratory

distinct

sues of the adult opossum

docu-

the

role

of

are not

muscle

smooth

in preparation)

nonsphincteric

clearly

reflects

significant

patterns

isoforms

gene expression muscle

segments.

differences

to

and

isoactins.

studies

of

segment

of y-smooth

the differential

et al., manuscript

flanking

path-

in the expression

The fact that similar likely

Distinct

rat gastrointestinal

isoactins

ways are not apparent tract

regulatory

actin

of such mechanisms

cytoplasmic

or cytoplasmic

ratios

bowel

patterns

by the fact that

of isoactin

isoactins

isoactins

and

that the composition

is supported

patterns muscle

muscle

in gastrointestinal

may vary from

in segment-specific

may help ciated

indicating

segment-specific

This observation

to a-smooth

exhave

tract is to establish

apparatus

machinery

these differential

gene that

smooth

of the

in this study suggest

segment.

examined.

isoactin mucosa,

role

cells.

the contractile

in

tissues

to the smooth musbowel each of these individual

primary

muscle

muscle

overlying

the contractile

smooth

in

are intrinsic

in the gastrointestinal

maintain

result

muscle

pat-

differences

segments

within

The

of the

observed

distinct

in bowel

of their

cle tissues found segments.

expression

segments

of smooth

that these differences

the

expression

in the smooth

are observed

makes

cell types irrelevant

gastrointestinal

sion in the functionally from

isoactin

gene expression

353

the segment-specific

tract reflect

isoactin

shown tinct

for the

in other

muscle

GI TRACT

observation

for this study. Consequently, the gastrointestinal

IN RAT

and y-cytoplasmic

coexpressed

isoactins

in all cell types

tract, including

smooth

are

of the mus-

cle cells where they may comprise as much as 30% 40% of the total isoactin mRNA pool.’ The ubiquitous nature

of these actin

isoforms

terpret

the

isoactin

gene expression

segment-specific

makes patterns

observed

it difficult

to in-

of cytoplasmic

in the adult rat gas-

various actin isoforms in the adult rat gastrointestinal tract. The y-smooth muscle and cl-smooth muscle isoactins are heterogeneously coexpressed in all of the

trointestinal tract. However, expression of the cytoplasmic isoactins appears to peak in those bowel segments possessing highly modified epithelia that would require complex cytoskeletal organization. This obser-

bowel segments examined in this study. In situ analysis (K. M. McHugh, manuscript in preparation) has shown that the smooth muscle isoactins are primarily coexpressed in the smooth muscle cells of the mature

vation is consistent with prior studies that have characterized the expression of the cytoplasmic isoactins in the mucosa of various bowel segments.26-28 The predominant cytoplasmic isoactin expressed in the adult

354

LIDDELL

ET AL.

GASTROENTEROLOGY

rat gastrointestinal

tract is the P-cytoplasmic

This observation mental

studies

isoactin form

is consistent that

with our earlier develop-

indicated

that

was the predominant

expressed

cytoplasmic

in most

isoactin

embryonic

gested a functional strating

alterations

of either

study. Therefore,

toplasmic

isoactin

differentiation types found The a-skeletal

of the

cum, colon,

rectum,

tectable

in the poly(A)+

testinal

segments.

nificantly Prior

studies”,12 skeletal

of the adult primarily

striated

muscle

similar

manner,

sphincteric this

would

skeletal

for

in these bowel muscle

a source

for

the

on the normal

and function isoforms

occurs

type(s)

found

tract,

that

some

within

the

suggesting

striated

muscle

Whether

isoactins

the

with

expression

an obvi-

detection segments.

of In a

in the

detection

histologically argue pressed

striated muscle isoactin in this bowel segment. Similar sources of skeletal muscle are not, however, known to exist in the cecum, colon, or rectum of the adult rat. More detailed histological analysis of these segments is currently underway in an attempt to clarify these issues. As with the smooth muscle isoactins, the primary function of the striated muscle isoactins appears to involve the establishment and maintenance of the contractile apparatus found in skeletal and cardiac muscle. It is postulated that the a-skeletal and a-car-

striated

studies

at the protein distinct

and

differentially

nature

muscle

expression

have

characterized muscle

the

isoactin

The identification

muscle

isoactins muscle

of

in the esophagus,

rat seems

to strongly

are being

level in these tissues. skeletal

of these

are not available.

of the adult muscle

in

level

of the striated skeletal

of our reisoactins

of the adult rat, simi-

and the heart.

that the striated

organs

level in the adult rat gastroinof the novel segments

distinct

protein

that the cytoplaswere

the differential

and anus

histologically of

isoactin

showed

at the protein

muscle

of

to be determined.

isoactins

of the

expression

in skeletal stomach,

Because

numerous

differential

patterns

of gastrointestinal

muscle

verifying

pat-

in the adult

in identical

remains

clearly

at the protein

However,

role for the

of this study are very consistent

studies

gastrointestinal

Skel-

gastrointestinal

observed

smooth

isoforms

cell

in these tissues.

in a variety

lar studies

actin

muscle

segment-specific

various actin

rat

studies 29-32 characterizing

These

of these

heterogeneous

the results

expression tissues.

tract. However,

functional

tract result

protein

prior

adult

the

development

gene expression

rat gastrointestinal However,

of skeletal to clarify

nonstriated

an expanded

the

that the rat cecum,

the expression

in

of the then

small quantities

of the rat gastrointestinal

it is also possible

detection

tissues of the anus. As with the esophagus, provide

muscle

ported

zone be-

is also present

be necessary

tract.

in stomach

provide the

therefore

role of this skeletal

indicate

smooth

hypotheses

as absolute,

It would

expressed

and

is taken

muscle

the

specificity

contain

testinal

analysis

of these

of this study would

studies.

its extent. found

isoactins

and sigof

both

and rectum

mic and

that it is composed

in the transition

muscle

the

expression

Histological

and stomach

isoactins

that

to the developing

muscle throughout

of skeletal

(data

from

If the tissue

No. 2

differences.

striated

results

isoactin ce-

is also de-

confirm

1 and 2. These observations

source

verified

that

shows

is even detected

studies

developmental

and heart.

rat esophagus

the

of these same gastroin-

is restricted

the esophagus

segments ous

stomach,

isoactins

suggested

muscle

of skeletal

etal muscle tween

earlier

have

these actin isoforms mature

have

muscle

fraction

our

that

the

distinct

However,

terns of isoactin

are heterogeneously

These observations

extend

cy-

of the cell

show

and anus. Additional

of the striated

in the

tract.

rat esophagus,

in our laboratory

expression

study

isoactin

are ob-

functional that

colon, muscle.

in the normal

of many

present

in the adult

muscle

of a precise

rat gastrointestinal

and a-cardiac

striated

examined

ratio may be essential

coexpressed not shown)

segments

and development

results

during

This hy-

ratios

maintenance

in the adult

to be verified.

that distinct

isoactin

served for most of the bowel

isoactins.

sug-

distinct

are functionally

muscle

have

morphology.

possess

it is assumed

remain

cells results

by the observation

isoactins addition,

the y-

the P-cytoplas-

in cultured

in cellular

P-cytoplasmic/y-cytoplasmic present

studies23

In

iso-

for this data by demon-

isoactin

is supported

whereas

diac

isoactins

actin

expressed

Recent

significance

mic and y-cytoplasmic in distinct

tissues,

was primarily

that overexpression

pothesis

the bcytoplasmic

cytoplasmic

adult

development.

isoactin.

Vol. 105.

ex-

In contrast,

has never

been

reported in the cecum, colon, or rectum of the adult rat. A histological review by our laboratory of random colonic segments from the rat, human, dog, and guinea pig is consistent with these results in not identifying any distinct skeletal muscle fibers within these organ segments (unpublished observation). Consequently, even though a-skeletal and a-cardiac isoactin mRNA is detected in these bowel segments, it is possible that the striated muscle isoactin proteins are not expressed. Similar types of ectopic expression of a variety of apparently tissue-specific transcripts have been reported studies are in several studies. 33,34 Clearly, additional

August

ISOACTIN

1993

needed to further characterize the precise nature of this expression in the adult rat gastrointestinal tract. In summary, the results of the present study show that the actin multigene family is heterogeneously coexpressed in a segment-specific manner in the adult rat gastrointestinal tract. In addition, distinct patterns of striated muscle isoactin gene expression are also observed in specified segments of the adult rat gastrointestinal tract. Both coordinate and independent regulation of isoactin gene expression is observed in the gastrointestinal tract, although distinct patterns of autoregulation are absent. These observations indicate that the mature gastrointestinal tract of the rat is highly complex and capable of segment-specific regulation of isoactin gene expression using a variety of molecular mechanisms. Whether this type of local regulation of gene expression results in, or is the product of, the distinct functional differences observed for the various bowel segments of the adult rat gastrointestinal tract remains to be determined. In conclusion, this study provides a basis for future studies designed to investigate the factors controlling the normal development and resulting diversity of the adult rat gastrointestinal tract.

tal expression

EXPRESSION

IN RAT

GI TRACT

of a smooth muscle y-actin in postmeiotic

355

male

germ cells of mice. 1989;9: 1875- 188 1.

10. Gunning P, Ponte P, Blau H, Kedes L. a-Skeletal and a-cardiac actin genes are coexpressed in adult human skeletal muscle and heart. Mol Cell Biol 1983;3: 1985- 1995.

11. Alonzo S, Garner I, Vandekerckhove

J, Buckingham

M. Genetic

analysis of the interaction between cardiac and skeletal actin gene expression in striated muscle of the mouse. J Mol Biol 1990;2 11:727-738. 12. McHugh K, Lessard J. The developmental expression of the rat a-vascular and y-enteric smooth muscle isoactins: isolation and characterization

of a rat y-enteric

1988;8:5224-523

actin cDNA. Mol Cell Biol

1.

13. Erba H, Gunning P, Kedes L. Nucleotide sequence of the human y-cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes. Nucleic Acids Res 1986; 14:52755294. 14. Gunning P, Ponte P, Okayama H, Engel S, Blau H, Kedes L. Isolation and characterization

of full-length cDNA clones for human a-,

p-, and y-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is sequentially removed. Mol Cell Biol 1983;3:787-795. 15. Chirgwin J, Pryzbla A, MacDonald R, Rutter W. Isolation of biologically active ribonucleotide acid from sources enriched in ribonucleases. Biochemistry 1979; 18:5294-5299. 16. Maniatis T, Fritsch E, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982;206. 17. Thomas P. Hybridization of denatured ments

transfered

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RNA and small DNA fragProc Natl Acad Sci USA

1980;77:5201-5205.

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Received August 18, 1992. Accepted April 5, 1993. Address requests for reprints to: Kirk M. McHugh, Ph.D., Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania 19107. Supported by National Institutes of Health grant HD27252. The authors thank J. Brittingham for assistance In this project.