EFFECTS OF CATECHOLAMINES AND ADRENERGIC BLOCKADE ON FLUID REABSORPTION IN ISOLATED RAT CAUDA EPIDIDYMIDIS

EFFECTS

OF

BLOCKADE

CATECHOLAMINES ON

FLUID

RAT

ADRENERGIC

REABSORPTION

CAUDA

P.Y.D.

AND

IN

ISOLATED

EPIDIDYMIDIS

WONG

and C.H.

YEUNG

Department of Physiology, Faculty of Medicine, University of Hong Kong, Hong Kong Accepted

Abstract-The rate measured in vitro.

September

19, 1977

of fluid reabsorption in the cauda epididymidis of rat has been Both adrenaline and isoprenaline produced a prompt, reversible

and dose dependent increase in the reabsorption rate. These effects were completely blocked by propranolol. The response to noradrenaline consisted of two components. n the presence of an alpha blocker, noradrenaline caused an increase, Iwhile in the presence of a beta blocker, it produced an inhibition in the rate of fluid reabsorption. The effects of these adrenergic agents were only observed when sodium ions were present in the intraluminal fluid, suggesting that they only affect the Na+-dependent component of fluid reabsorption. The possibility that they may affect the active transport of sodium in the duct was discussed. The effects of these adrenergic agents were inter preted in terms of the presence cauda epididymidis.

of alpha and beta receptors

in the epithelium

of the rat

It is now well established that fluid reabsorption takes place along the entire length of the mammalian epididymal duct.

A major part of the rete testis fluid is reabsorbed as it

flows down the epididymis (1, 2, 3). The mechanism of fluid reabsorption has been studied in isolated duct of rat epididymis in vitro. It was found that 50 % of the fluid reabsorption is secondary to an active transepithelial transport of sodium ions (4, 5).

Furthermore this

process, like many epididymal functions, is abolished by castration, indicating that the fluid reabsorption process is dependent upon the presence of circulating androgens (6). There is little information

on the role of neurotransmitters

in the functions of the

epididymis, although it has been shown that the cauda epididymidis is heavily innervated by sympathetic and parasympathetic

nerves (7, 8). In this paper, we report the effects of

adrenergic agents and blockers on fluid reabsorption

in isolated rat cauda epididymidis.

The results were interpreted in terms of the presence of the alpha and beta adrenergic re ceptors in the epididymal epithelium. MATERIALS Male Sprague-Dawley the head. solution.

The epididymis A segment

fat and connective for measuring

onto the platform,

rats weighing

between

200-300

was quickly

removed

and placed

of the cauda

epididymidis

tissue and placed

secretory

AND METHODS

rate in isolated

(about

on a specially

by a blow on

in cold Kreb's

bicarbonate

0.3 cm long) was dissected

designed

rat seminiferous

g were sacrificed

platform

tubules (9).

free of

similar to that used Each end was clamped

and incisions were made in the duct close to the clamps.

The lumen was

then flushed with Kreb's bicarbonate solution to remove all the spermatozoa.

After closing

one end with a silk ligature, the tubule was filled with about 0.25 ,al Kreb's bicarbonate solution, using a polyethylene cannula pulled to a tip diameter of about 150 ,1m, and the other end of the segment was also tied.

The whole operation was performed under a

dissecting microscope at x 16 magnification.

The sac of epididymal duct (about 1-2 mm

long), supported on the platform, was then placed in a small bath (volume 10 ml) filled with normal Kreb's bicarbonate solution which was bubbled continuously with 5 %C02 in 02. The bathing solution was maintained

at 35+0.5'C

(reabsorption

rate was maximal at

35'C) using a glass heat-exchanger immersed in the bath and the temperature was monitored by a thermistor probe.

The peritubular and intraluminal fluids were always iso-osonitic.

For estimation of the rate of fluid reabsorption, the bath was examined under x 40 magnification and the internal diameter of the lumen was measured at intervals of 0.4 mm along the length of the sac, using an eyepiece micrometer.

The mean luminal diameter

of the duct was obtained and, by knowing the length of the sac, the luminal volume was calculated.

After a 10-min equilibration period, readings of the diameters were taken at

10-min intervals. in the duct.

A reduction in the luminal volume indicated a net reabsorption of fluid

The rate of fluid reabsorption

tubule in 10 or 30 min.

was expressed in of fluid reabsorbed/cm2 of

The 'reabsorptive area" was calculated from the mean internal

radius of the tubule and its length (2 7rrl). The Kreb's bicarbonate solution used had the following composition (mM): NaCI, 118; KCI, 4.7; CaC12i 2.56; MgSO4, 1.13; NaH2PO4, 1.17; NaHCO3, 25; glucose, 11.1. When gassed with 5 % CO2 in 02, it had a pH of 7.4. When sodium-free solution was used such was substituted for by an equivalent amount of choline and the NaHCO3 by KHCO3 (5.9 mM). In this instance, KCI was omitted and the solution was bubbled with pure 02 to obtain pH 7.4. The experimental protocol was as follows: fluid reabsorption

was measured over a

30 min control period followed by the addition of adrenergic agents.

Fluid reabsorption

was then taken for a further 30 min period to characterize the effect of these agents. most cases, the epididymal ducts were washed with normal Kreb's bicarbonate again and fluid reabsorption was followed for a further 20 min.

In

solution

When blockers were used,

they were added to the bath 2 min before addition of the adrenergic agents. The following drugs were used: 1-adrenaline hydrogen tartrate (BDH); dl-isoproterenol sulfate (Sigma); 1-norepinephrine

bitartrate

(Sigma); dl-propranolol

HCl (Sigma); and

phenoxybenzamine (Smith, Kline & French) in 0.03 % propylene glycol. They were prepared 1-3 min before administration and were added to the peritubular fluid only. RESULTS Effects of adrenergic agents on fluid reabsorption The effects of adrenaline applied to the peritubular fluid is shown in Fig. I Adrenaline (10-5 M) produced a prompt and sustained increase in the fluid reabsorption rate. This effect of adrenaline was reversible on washing. The increase in fluid reabsorption was

FIG. 1. Effect of adrenaline (10-5 M) (C~) and adrenaline (10-' M) (0) on the rate of fluid reabsorption in isolated duct of rat cauda epididymidis. (C) control rate. Each point represents the mean-CS.E. from (C) six experiments, except for the points where the number of experiments are shown in parentheses; (®) nine experiments and (C) six experiments. The drugs were applied to the peritubular fluid at the first arrow and were washed out of the bath at the second arrow.

FIG. 2. Effect of propranolol (10-' M) ('j) and propranolol (10-5 M) (•) on the response to adrenaline (10 M). (P) control rate. Each point represents the mean j S.E. from (. ) five experiments; (•) five experiments and (C;) six experi ments. The drugs were applied to the peritubular fluid at the first arrow and were washed out of the bath at the second arrow.

estimated by calculating the percentage increase in rate compared to the control. experiments, adrenaline (10-s M) caused a 102.2--17.8% (meanCS.E.M.)

In six

increase in the

rate of fluid reabsorption over periods of 30 min following addition of the drug (P<0.001). A lower dose of adrenaline (10-q M) caused 57.8±7.9%

(mean C S.E.M.) (5 experiments)

increase (P<0.001). When the duct was pretreated

with propranolol

(10-q M), the stimulating

effect of

adrenaline (10-7M)

was reduced (Fig. 2). The increase in the rate of fluid reabsorption

over the control was 19.0+12.5% significant at P<0.05).

(fluid reabsorption

during the period 30-40 min was

When a higher dose of propranolol (10-5 M) was used, the stimu

lating effect of adrenaline (10-7M) was completely suppressed.

Under this condition, the

rate of fluid reabsorption was found to be reduced by 19.2±-9.4% (mean±S.E.M.) this effect was not significant statistically.

although

These results suggest that the effect of adrenaline

on fluid reabsorption was mediated through a beta action. by adrenaline after beta blockade (propranolol,

The slight inhibition produced

10-5 M) may be attributed to the alpha

action of the drug. The effect of isoprenaline on the rate of fluid reabsorption is shown in Fig. 3.

Iso

prenaline (10-5 M) caused a stimulation of fluid reabsorption which is similar to that seen with adrenaline. The increase was 50.9±9.4%%(mean ±S.E.M.(P<0.001) (9 experiments). A lower dose of isoprenaline (10-7M) also stimulated fluid reabsorption

by 27.5±16.2%

(mean±S.E.M.) but the result was not significantly different from the control. Fig. 4 shows a representative experiment in which the duct was treated with propranolol (10-5 M) prior to addition of isoprenaline (10-5 M). This concentration of propranolol completely blocked the effect of isoprenaline. However, on washing out the antagonist, the same concentration

of isoprenaline produced a large increase in the reabsorption rate.

The effects of noradrenaline

and blockers are shown in Fig. 5. Addition of nor

adrenaline (10_6 M) alone had no significant effect on the rate of fluid reabsorption. However, pretreatment of the epididymal ducts with phenoxybenzamine (10-5 M) prior to addition of noradrenaline (10-6 M) stimulated reabsorption rate by 44.6±7.5% (mean±S.E.M.) (P<0.005) (5 experiments).

On the other hand, if the ducts were pretreated with propranolol

(10-7M), no effect on the rate of fluid reabsorption was seen. However when the dosage of propranolol was increased to 10-5 M, an inhibition caused by noradrenaline (10-7 M)

Ft(,. 3. Effect of isoprenaline (10-5 M) (0) and isoprenaline (10-7 M) (S) on the rate of fluid reabsorption in isolated duct of rat cauda epididymidis. Each point represents the mean±S.E. from (C) nine experiments and (0) four experiments. Isoprenaline was applied to the peritubular fluid at the first arrow and was washed out of the bath at the second arrow.

FIG. 4. Effect of stimulation of beta receptors on the rate of fluid reabsorption crossing one isolated duct of rat epididymis. The first arrow indicates the addition of propranolol (10-6 M) to the peritubular fluid. This antagonist completely blocked the effect of isoprenaline (10-5 M) added at the second arrow. On washing out the antagonist (third arrow), isoprenaline (10-5 M) elicited a large increase in the reabsorption rate (fourth arrow). The beta stimulant was washed out of the medium at the fifth arrow.

FIG. 5. Effect of noradrenaline (10-6 M) (C); noradrenaline (10-6 M) in the presence of phenoxybenzamine (10 M) (0); noradrenaline (l0-' M) in the presence of propranolol (10-5 M) (C) and noradrenaline (10-' M) in the presence of propranolol (10-6 M) (A) on the rate of fluid reabsorption in the isolated duct of the rat cauda epididymidis. Each point gives the mean+S.E. from (0) six experiments; (0) five experiments; (C) five experiments and (A) five experiments. The drugs were added to the peritubular medium at the first arrow and were washed out of the bath at the second arrow.

was obtained.

The decrease in rate over the control was 45.6C7.6 % (mean+S.E.M.)

(P<0.005) (5 experiments). Effect of intraluniinal In attempts

sodium ion removal on the responses to adrenergic

to show whether

the active sodium transport

the effects of adrenergic

mechanism,

agents

agents were mediated

through

the effects of these agents were studied in the isolated

ducts deprived of intraluminal ions.

sodium

In these experiments, the lumens

of the ducts were filled with a sodium free solution (sodium was substituted by an equivalent

amount

of choline, and

NaHCO3 by KHCO3) and the tubules were

incubated

containing

in a normal

solution.

sodium

Fig. 6 shows the

results from these experiments. reported

previously

As was

(4), when sodium

was removed from the intraluminal fluid, fluid transfer

was inhibited

by 50%.

The effects of adrenergic agents on fluid reabsorption

in the presence of intra

FIG. 6. Effect of removal of intraluminal sodium ions on the rate of fluid reabsorp tion in the isolated duct of rat cauda epididymidis. Reabsorption rate was ex pressed over 30 min. The columns on the left show the effect of adrenaline (10-5 M) (B) and noradrenaline (10-' M) + pro pranolol (10_, M) (C) on the rate of fluid reabsorption when the duct was filled with normal sodium-containing solution. (A) control. The columns on the right show the responses to these catecholamines in the absence of intraluminal sodium ions. (A) control; (B) adrenaline (10 M) and (C) noradrenaline (10-7 M) + pro pranolol (10-6 M). Each column shows the mean }_S.E. The number of experi ments is shown in parentheses.

luminal sodium were taken from previous experiments except the rate was expressed over 30 min. Adrenaline (l0-s M) caused a 102% increase in the rate of fluid re absorption (P<0.001) and noradrenaline (10_7 M) in the presence of propranolol (10-s M) caused a 45.6 % decrease in the reabsorption rate (P<0.005). However, when the intraluminal

sodium was re

placed by choline, no effects of these adrenergic agents were observed.

DISCUSSION Although

El-Badawi

& Schenk (7) and Norberg,

that the cauda epididymidis and adrenergic of vasomotor the muscle terminate

of many mammalian

nerves, the exact physiological and myomotor

coat in variable and form

tone (10, 11), has not been elucidated.

These nerves penetrate

numbers

a loose

and to different

intra-epithelial

water

In

the

Adrenaline epididymis.

pretreatment

this

rat cauda

and isoprenaline

by the beta

bath,

no significant

of the epididymis

depths,

and in many

in the epididymidis.

function

paper,

stimulated

action

network

in regulating

we demonstrated

epididymidis

These effects could be blocked

was mediated incubating

epithelium.

in the isolated

by cholinergic

apart from their control

an important

across

(8) have shown

role of these nerves,

that these nerves may subserve

reabsorption

Risley & Ungerstedt

species are heavily innervated

is regulated

by propranolol,

of the drugs.

When

effect on the reabsorption with phenoxybenzamine

suggesting

of ions and

the rate

by adrenergic

the rate of fluid reabsorption

they

It is possible

transport that

species,

of fluid

agents.

in the isolated

rat

that the stimulation

noradrenaline

was added

rate was observed. prior to addition

to the

However,

of noradrenaline

stimulated the rate of fluid reabsorption

by 45 %.

the presence of a beta blocker, propranolol,

On the other hand, noradrenaline, in

depressed reabsorption

rate by 46 %.

The

former effect may be attributed to the beta action of the drug, while the latter response to may be due its alpha action.

The effect of noradrenaline alone would be the net result of

opposing actions of both alpha and beta components of this catecholamine. The responses to these adrenergic agents on fluid reabsorption may have resulted from contraction of the muscle layers caused by these agents.

However, we consider this event

to be rather unlikely since responses in fluid reabsorption

to these adrenergic agents were

dependent on the presence of sodium ions in the intraluminal fluid medium.

When all the

sodium ions were removed from the intraluminal medium, the fluid reabsorption rate was reduced by 50%.

A similar degree of inhibition was obtained when amiloride (10-4 M)

was added to the luminal surface of the epithelium.

The result was taken to indicate that

50% of fluid transfer was secondary to an active transepithelial transport of sodium (4). When the effects of these adrenergic agents were studied in the absence of a transepithelial sodium transport (i.e. in the absence of intraluminal sodium), the stimulating effect of adrenaline and the inhibitory effect of noradrenaline were not seen. that the effects of adrenergic agents on fluid reabsorption

It is pertinent, therefore,

were mediated through their

actions on the sodium transport process. There is evidence that sympathetic agents regulate sodium transport in many epithelia. Martin & Young (12), Schneyer & Thavornthon (13) and Schneyer (14) when studying the electrolyte transport in perfused salivary duct found that alpha adrenergic agents caused a decrease while beta agents caused a rise in the rate of sodium reabsorption Furthermore,

in the duct.

Greven and Heidenreich (15) performed micropuncture on rat kidney and

suggested that beta agonists increased sodium and water reabsorption in the distal nephron. Our results are therefore consistent with the effects of adrenergic agents on sodium transport in these tissues.

It is of interest to note that transport processes in the rat cauda epididymidis

are very similar to that occurring in the distal tubule of the kidney. Using a microperfusion technique, we have recently demonstrated that the rat cauda epididymidis reabsorbs sodium and water and secretes potassium (16). transepithelial transport in frog skin.

Sympathetic agents were also found to affect

Alpha stimulants reduce net Na+ flux (17) and beta

stimulants enhance it (18, 19). The exact mechanism underlying the actions of these adrenergic agents on sodium transport hence fluid reabsorption in the rat cauda epididymidis is uncertain.

Cyclic AMP

has been shown to be the mediator of adrenaline-induced activation of liver phosphorylase and has been proposed as the mediator of the action of catecholamines on other systems (20). It is conceivable that beta adrenergic agents stimulate the generation of intracellular cyclic AMP in the epididymal epithelium, while alpha agents decrease it (21). In a separate study, when the luminal membrane of the rat cauda epididymidis was impaled with micro electrodes, it was found that it has a high permeability to sodium ions (22).

Adrenaline

(10-7M) reversibly depolarized this membrane. The final effect of adrenaline seemed to be due to an alternation in the sodium conductance of the luminal surface of the epithelial

cells (unpublished). With the present data, it can be concluded that both alpha and beta adrenergic receptors may be present in the epididymal epithelium.

Beta receptor activation produces a rise while

alpha receptor stimulation produces a fall in the rate of fluid reabsorption. are dependent on the presence of intraluminal sodium ions.

These effects

Whether these receptors play

a physiological role in the control of fluid reabsorption in the cauda epididymidis remains an open question. Acknowledgement:

This work was supported by a grant from the World Health

Organization.

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