Effects of Phenylephrine, Butylsympatol, Pilocarpine, and Lithium on Runout of Rb from Isolated Rabbit Ciliary Processes

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Effects of phenylephrine, butylsympatol, pilocarpine, and lithium on runout of 86Rb from isolated rabbit ciliary processes

Janet Vale'6 and Ernst H. Bdrdny

Isolated ciliary processes from pigmented rabbits were incubated with S6Rb, washed, and transferred to a runout medium. The appearance of 8GRb in this medium was followed. Uptake of seRb is decreased by lack of glucose and/or oxygen, and by 10 times excess potassium. Runout is decreased by cold (+10° C.) and by 3 or 30 mM. Ca and increased by complete substitution of lithium for sodium. Pilocarpine (10~3, 10~5M) had a small accelerating effect limited to the initial phase of runout, while the stable /3-adrenergic agent butylsympatol (10~sM) in the presence of 2 mM. Ca had a very small but more longlasting accelerating effect. The a-adrenergic phenylephrine (10~2M) had no effect.

Key words: ciliary processes, rubidium isotopes, biological transport, potassium, pharmacodynamics, pilocarpine, phenylephrine, orciprenaline, butylsympatol.

T-he effect of autonomic drugs on aque- of ciliary processes of the rabbit have indi- ouJLs hhumor secretion is difficult to study in cated inhibitory effects on rate of shrink- the intact eye since the drugs affect circu- age (interpreted as secretion) by pilocar- lation and outflow facility. Studies in vitro pine as well as by adrenaline.1 The anion pump of Becker- and others is also inhibited by pilocarpine at high concentrations although a weak stimulatory effect 3 From the Department of Pharmacology, University was seen at lower concentrations. A simi- of Uppsala, Uppsala, Sweden. lar effect on amino acid transport has also 4 This study was supported by Research Grants been found. This was shown by demecari- B67-14X-733-03B from the Swedish Medical um but not by echothiophate and hardly Research Council, and NB-3637-05 from the by pilocarpine. Thus, certain effects of Institute of Neurological Diseases and Blindness, autonomic drugs seem to exist. However, United States Public Health Service, Bethesda, Md. whether these are due to anything similar Reprint requests to Ernst H. Barany. to excitation of gland cells by such drugs Manuscript submitted Sept. 9, 1968; revised is not at all clear. manuscript accepted Nov. 5, 1968. Histological evidence has shown no "Present address: Department of Pharmacology, adrenergic fibers in the epithelium proper University of Manchester, England, of the ciliary processes, but a subepithelial 422

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plexus exists.5'G Because of the stability of out distinguishing the different kinds1"4- 15) were the transmitter, this would not prevent dissected free from the iris under the dissecting microscope and placed in small nylon mesh effects of sympathetic nerves on the func- pouches. Circular pieces of nylon mesh, approxi- tion of the epithelium. Direct cholinergic mately 2 cm. in diameter, were cut from stock- innervation of the epithelium has not been ings. A cotton filament was threaded along the found, but again a subepithelial plexus periphery and a loose knot formed. After the exists.0- 7 The lack of direct innervation processes had been put in place at the center of the piece of mesh, the knot was tightened so that does not exclude receptors. a small pouch was formed. There was ample The present paper is an attempt to test space in the pouch. Generally the processes from whether autonomic drugs excite the cells each eye were divided into 2 or 3 portions of of the ciliary processes in a similar manner about 5 mg. each. The small heap of tissue frag- as they excite certain other gland cells. ments could not be blotted and weighed reliably. 8 When 2 portions of ciliary processes were obtained Burgen showed that the submaxillary from each eye, one of these became a control gland loses potassium to the blood, as well preparation and the other experimental. When 3 as to the saliva, when stimulated in vivo. portions were obtained, they were divided such This has been further analyzed by Coats, that 2 from one eye and 1 from the other were Denton, and Wright9 in the sheep parotid used as control and similarly for the experimental preparations. The distribution was at random. gland. Schneyer and Schneyer10 studied Loading. No systematic study of the uptake the washout of radioactive potassium from time course was made. The pouches with the slices of rat submaxillary gland. An in- ciliary processes were incubated for 30 minutes crease in the rate of loss and uptake was in 3 ml. of incubation fluid containing approxi- caused by pilocarpine (1.2 x 1O~5M), and mately 1 /J-C of SGRb. The concentration of stable this effect was prevented by atropine (2.4 rubidium varied but was below 1 mM. The fluid 5 was contained in small conical flasks (25 ml. with x 10~ M). The present experiments were a bottom diameter of 4 cm.) and up to 3 pouches designed similarly. simply dropped in. Pure oxygen was led in For practical reasons SGRb was used in through a loose-fitting cap to each bottle. The the present study instead of 42K. Burgen bottles were shaken in a shaking incubator (100 and Spero11 have found that the stimula- cycles per minute, peak to peak amplitude 4 cm.). Standard temperature was 30° C. tion of smooth muscle with cholinergic Washing. At the end of the loading period the drugs greatly increases the rate of loss of pouches were picked up and transferred to S(iRb, and we hoped this to be true in the bottles containing 10 ml. of the incubation fluid present system also. It must be remem- maintained under the same conditions (but lack- bered, however, that Rb is not an ideal ing rubidium) as the fluid in the loading bottles 12 13 but not gassed except when expressly stated. The tracer for potassium. ' pouches were shaken for 30 seconds in each of 2 successive bottles. The pouches were blotted be- Methods and materials fore transfer to the runout bottles. Pigmented rabbits, male and female, were used. Runout. The runout bottles (5 ml. with a bot- Hay, oats, and water were available ad libitum. tom diameter of 2.5 cm.) contained 3 ml. of fluid In some of the earlier experiments the rabbits and were gassed and shaken in the same manner were anesthetized with 1.5 Gm. per kilogram in- as the loading bottles. Standard temperature again travenous urethan (25 per cent) and one eye was was 30° C. 50 /*L samples of the medium, the removed for immediate use. The animals were "runout fluid," were removed from the bottles kept warm with heating pads and killed by air with constriction pipettes. Each runout bottle had emboli some 2 hours later. The second eye was its own pipette which was used throughout the then removed and treated as the first. In the re- whole period. Sampling times were 2, 4, 6, 8, 10, mainder of the experiments the animals were an- 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, and 60 esthetized with intravenous urethan and killed minutes after the pouch was transferred to the with air emboli. Both eyes were then removed at runout fluid. Two samples were taken on each of the same time. After excision of the eye the iris the last 4 sampling occasions. The runout bottles and ciliary body were removed and placed flat were then transferred to a beaker of boiling water on plastic-covered ice. During the dissection the in order to kill the preparations. Finally 5 samples tissues were kept moist with incubation fluid. With of runout fluid were removed. the use of fine scissors the ciliary processes (with- Plating and counting. The samples wore blown

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onto pieces of filter paper fitted into the bottom sulfate (Alupent, Boehringer Ingelheim); phenyl- of 1 inch planchets. The papers were allowed to ephrine hydrochloride; pilocarpine hydrochloride; dry and then counted in a Tracerlab counting sys- propranolol hydrochloride (Inderal I.C.I.). tem using a G.M.-tube with a window of 2.8 mg. per square centimeter. Preset counts were Results used and 3,000 counts usually accumulated. Con- Uptake experiments. trol experiments showed that no correction for Accumulation ratio. In a few experiments coincidence was necessary. conducted for other purposes the ciliary Mathematics. From the counts present in the runout fluid after killing the tissue and the total processes were allowed to remain on the amount present in the individual samples removed iris during uptake. They were then dis- for counting, one obtained the total amount of sected, blotted, and weighed and the radio- counts present in the tissue (and pouch) at the activity measured. The ratio tissue/medium start of the runout period. Thus, from the amount for Rb was 15 to 20. removed with the previous samples and the amount present in the runout fluid, the total Glucose. Absence of glucose reduced the amount of activity remaining in the tissue at each uptake at 30° C. whether or not oxygen sampling time could be estimated. Since some was present. In 5 control experiments with moisture adheres to the pouch and, moreover, be- glucose the average total activity in the cause the volume of the boiled tissue adds to the tissue was 5,860 c.p.m.; in the parallel ex- distribution volume, a small correction was intro- duced; it was assumed that these 2 volumes periments without glucose, with ciliary amounted to 30 /*L when the total amount of processes from the same eyes as the con- counts in the runout fluid after killing was esti- trol experiments, it was 4,090 c.p.m., or mated. 30 per cent less. The tissue samples had Because of the removal of a total of 0.9 ml. not been weighed, but random differences with the samples the volume against which the should occur equally in both directions. runout took place decreased from 3.0 ml to 2.1 ml. during the experiment. If the runout were a Oxygen. In 9 experiments gassed with simple exponential in a bath of infinite volume, it oxygen at 30° C. the average total activity would cease to be so in a decreasing bath. How- in the tissue was 10,110 c.p.m. In parallel ever, even at the end of the runout period some experiments with ciliary processes from the 10 to 20 per cent of the initial activity were still present in the tissue. Therefore, the tissue concen- same eyes as the control experiments, with tration was roughly 50 to 100 times the concentra- the oxygen replaced by nitrogen the aver- tion in the medium and the error caused by de- age total activity was 7,430 c.p.m. or 26 creasing medium volume negligible. The results per cent less. The tissue samples had not can therefore be plotted on a semilogarithmic been weighed. scale. Since exactly the same schedule was kept Glucose and oxygen. In 6, experiments throughout, corresponding runs were pooled by at 30° C. glucose was omitted from the averaging the per cent activity remaining in the incubation fluid, and the oxygen was re- tissue at each time. When the t test was used, it placed by nitrogen. The average total ac- was applied to one point at a time. P values refer tivity in the tissue was 16,000 c.p.m. in the to one point; no attempt was made to pool infor- mation from several points. control experiments, while in the 6 parallel Solutions. The standard solution for incubation experiments, with ciliary processes from and runout was the same as that used by Berg- the same eyes, but deprived of glucose gren1G and had the following composition in milli- and oxygen it was 5,960 c.p.m., 63 per cent moles per liter: NaCl, 150; KC1, 3; MgSo,, 1; less, or roughly the sum of the individual glucose, 7; tris (tris [hydroxymethyl]aminometh- ; ane), 15.4. The tris was added (310 ml. per liter) effects of the lack of O2 and glucose. in the form of a tris-HCl buffer, pH 7.4, con- Excess potassium. The standard incuba- taining 6.05 Gm. of tris per liter. When composition fluid contained 3 mM. K. If the Rb tion was altered, osmolarity was corrected with in fact acted as a substitute for K, its up- NaCl. Analytical grade chemicals were used take should be reduced by excess K, even throughout. Drugs. Drugs used were: acetazolamide sodium if one cannot expect simple equivalence. (Diamox, Lederle Laboratories); butylsympatol In 4 experiments with 10 times the con- (Vascular, Boehringer Ingelheim); orciprenaline centration of K in the incubation fluid the

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about the last 3 points (40, 50, and 60 minutes) could be due to decay of the tissues. However, later experiments re- vealed that at least some of this bending could be due to insuflBcient heating of the tissue at the end, so that not all the activity was released into the fluid. The result would be an underestimation of the amount of activity in the tissues. This effect, while negligible in the early stages, would be- come more prominent in the later stages and bend the curve. An opposite effect is caused by the activity adhering to the tissues and pouch when first placed in the 0 10 20 30 40 50 60 MIN SG runout bottle. Thus, we place little empha- Fig. 1. Rabbit ciliary processes. Runout of Rb sis on the second half of the curves. into standard solution. 30° C. Gassed with oxygen. Abscissa: time in minutes in runout solution. Ordi- REPRODUCIBILITY. As mentioned under nates: Per cent of total activity remaining in tissue. Methods, the ciliary processes from one Mean of 10 experiments with one tissue sample in eye were divided into 2, and sometimes 3, each. portions. Individual curves indicate that the different preparations from the same eye generally behaved in a similar manner uptake of Rb was reduced by 60 per cent. as shown in Fig. 2, A. Differences were In these experiments the chemical concen- occasionally seen as in Fig. 2, B. Prepara- tration of Rb was roughly 7 x 10~2 mM. tions from the 2 eyes of the same rabbit Runout experiments. also generally showed close agreement (as General shape of the runout curve. An do 3 of the 4 curves in Fig. 2) even when example of the runout curve is shown in the second eye was removed and dissected Fig. 1. The line does not extrapolate to some 2 hours after the first. Differences 100 per cent at time zero. This indicates which did occur bore no systematic rela- that some activity is washed out into the tionship to the order of utilization of the runout fluid very quickly. It is not possi- eyes. The greatest differences were ob- ble to say whether part of this is extracellu- served between different rabbits; the differ- lar activity or if all is simply incubation ences appeared to be unrelated to the sex fluid adhering to the pouch despite the or size of the rabbits or the season of the washing and blotting. The graph shows year. Because of the sometimes erratic be- a faster initial drop, then becomes an al- havior, at least 6 preparations were used most straight line, with a tendency for the as a rule for averaging. "tail" of the curve to turn down. A conven- A sudden downward step, like the one tional graphical analysis into several phases seen in one curve of Fig. 2, B, could be with different half-lives was tried. There due to a small but active tissue fragment are, however, several sources of error which had slipped through the nylon mesh which tend to invalidate the results: The and been picked up in the 50 /xL sample. amount of external fluid adhering is not In Fig. 2, B, this was not the case; the known, the geometry of the several tissue second sample was not especially high, but fragments in the pouch is not identical, the first was low. and the amount of tissue crushed can vary. Conditions during runout. We, therefore, refrained from using the TEMPERATURE. To make these experi- half-time analysis. The tendency for the ments comparable with those of Berggren, tail of the curve to turn down especially a standard temperature of 30° C. was

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"/. in 100 TISSUE 80ro ". B 60

0 o 20 0

10 20 30 40 50 60 MIN 10 20 30 40 50 60 MIN Fig. 2. Runout of SGRb into standard solution. Duplicate experiments in 2 eyes of the same rabbit. The 2 preparations in A came from the same eye, the two in B from the contralateral one. The eye, used for the A curves, was removed from the anesthetized rabbit 2 hours later than that used for the B curves. Conditions as in Fig. 1.

V. in 100 TISSUE 80 B \ 60 V sN *• \» - 40 0 • • 10*C X> n-3 x • 38 'C • 45°C ^^^^ 20 s n = 10 N N \ N N s Nss30°C Ss 30*C > 10-

0 10 20 30 40 50 60 MIN 1 1 i i 10 20 30 40 50 60 MIN 10 20 30 40 50 60 MIN Fig. 3. Effects of temperature on runout of SGRb. The 3 broken lines are for reference and are derived from the data of Fig. 1, run at 30° C. The runs at different temperatures were done on different occasions. Therefore, n in this figure means number of tissue samples run. Standard solution, oxygen.

chosen for the incubation and runout. ferent temperatures. The standard curve However, a few experiments were per- for 30° C. was obtained earlier. The results formed at different temperatures. In 3 ani- are shown in Fig. 3. Runout is somewhat mals (5 preparations) both incubation and faster at 30° C. This agrees with Berg- runout were made at 38° C, in 5 animals gren's findings of an optimum around 30° (10 preparations) both were at 45° C, C.1T Runout at 10° C. is markedly slower. while in 2 animals (3 preparations) incu- OXYGEN. Oxygen was replaced by nitro- bation was at 30° C. and the runout at gen during incubation and runout while 10° C. Different animals were used at dif- the controls had oxygen throughout. In

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•/. in TISSUE 100 80 o B 60 o °ooo o o

n- 6 No O2 • No O2 No GLUCOSE 10 20 30 40 50 60 MIN 0 10 20 30 40 50 60 MIN Fig. 4. Effect of oxygen lack alone (A) or combined with glucose lack (B) on runout of SGRb. 30° C. Controls (open circles) were run in standard solution, with oxygen. Conditions were the same during loading with Rb, washing, and runout. Number of pairs of experimental and control tissue indicated by n.

these series, even the washing fluid was TENFOLD EXCESS POTASSIUM DURING LOAD- gassed with N2 or O2. A small effect was ING AND RUNOUT. For the reasons just men- seen, Fig. 4, A. When both oxygen and tioned one would expect the excess K glucose were lacking, Fig. 4, B, during curve to run below the control since excess incubation and runout (the controls having K reduces the uptake during loading. This both throughout), the effect was perhaps is indeed the case; the experimental curve more marked. The main effect seems to runs some 15 to 20 per cent below the be that the experimental curve runs below control (n = 4). the control one. Since they are both ex- EXCESS POTASSIUM DURING RUNOUT ONLY. pressed in per cent of the original content If incubation is performed with normal K of the tissue, the explanation could be and only the runout is done with 10 times rather trivial. There has to be a very rapid excess K, there is at most a minimal in- initial loss of activity to cause this result. crease in the rate of runout and only dur- Part of this initial loss is undoubtedly due ing the first 15 minutes (n = 4). Berg- to loss of physically adhering activity. gren10 also found no effect. Since, as mentioned, the effect of combined LACK OF POTASSIUM DURING RUNOUT ONLY. glucose and oxygen lack reduces the up- The no-K curve runs about 20 per cent take of Rb by more than half, physically below the control, Fig. 5, A. The difference adhering activity would have a greater ef- is statistically significant at p < 0.01 tested fect in these experiments, and explain the at minutes 2 and 10. The shapes are not greater initial runout. Whether there are very different but there seems to be a other factors also involved cannot be de- somewhat faster initial part. We have no cided. explanation for this finding. Using a differ- 17 GLUCOSE. Absence of glucose had no dis- ent buffer, Berggren found shrinkage of cernible effect (5 pairs of tissue samples the processes retarded in a K-free medium. compared, i.e., n = 5). To achieve sub- REPLACEMENT OF SODIUM BY LITHIUM 10 strate depletion in these experiments, even DURING RUNOUT ONLY. Berggren found a the incubation and washing had been done complete inhibition of the shrinkage pro- without glucose while the control experi- cess with high lithium concentration. In ments had glucose throughout. the present experiments, however, lithium

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7. in 100 . TISSUE 80 b •°o_ A «o B 60

40 H._ o

n= 9 n=6 • 150 mM Li 10 No K No Na

10 20 30 40 50 60 MIN 10 20 30 40 50 60 MIN Fig. 5. Effect of potassium lack (A) or of replacing sodium with lithium (B) on runout of SGRb. Controls (open circles) were run in standard solution. 30° C, oxygen. Number of pairs of experimental and control tissue indicated by n.

7. in TISSUE 100 80 B •§e A 60 c •> o o • 40 o • o o • o - o ° • o o n-6 20- 6 30 mM Ca 8 n-7 3 mMCa No K 10-

0 10 20 30 40 50 60 MIN 10 20 30 40 50 60 MIN SG Fig. 6. Effects of calcium ion on runout of Rb. In A, 3 mM. CaCl2 was substituted for 3 mM. KC1. In B, 30 mM. CaCl2 was substituted for 30 mM. NaCl. Controls (open circles) were run in standard solution. 30° C, oxygen. Number of pairs of experimental and control tissue indicated by n.

accelerates the runout during a first phase 3 mM. K, there was a conspicuous ab- of about 15 minutes while it seems to have sence of any rapid initial phase in the con- no effect on the further course, Fig. 5, B. trol curve, see for instance Fig. 7, C. CALCIUM DURING RUNOUT ONLY. A high ACETAZOLAMIDE DURING RUNOUT ONLY. At concentration of calcium (30 mM.) slows the pH used (7.4) there was no effect at down the initial phase of the runout mark- a concentration of 10~5M. This is in agree- edly. In the absence of potassium even a ment with Berggren.17 moderate amount of calcium (3 mM.) has Autonomic drugs. It seemed of interest a similar effect, Fig. 6. In 3 groups of to compare the action of autonomic drugs experiments with drugs (n = 6 in each), under the same conditions as Berggren,1 where 2 mM. Ca was present together with i.e., with no calcium in the incubation and

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% in 100 TISSUE 80 8 A i B i C : 2°o^# o o • 60 ••2 o o o •• o m • o •• o 8 • o • • o • 40 - • o o • • o • ° n=6 • • o • o * imM • o n- 6 - c BUTYL- • * 11 — D 10" mM i r SYMPATOL 20 PILO 1 MI ff PILO

10 -

0 10 20 30 40 50 60 s I 0 10 20 30 40 50 60/0 10 20 30 40 50 60 MIN Fig. 7. Effects of pilocarpine and of butylsympatol on runout of SGRb. In A and B, pilocarpine was used in the standard solution and neither controls nor experimentals had any calcium. In C, butylsympatol was used, and both controls and experimentals contained 2 mM. Ca, 30° C, oxygen. Number of pairs of experimentals and controls indicated by n.

runout fluid. However, since calcium has Phenylephrine (Neosynephrine). This been shown to be essential for the stimu- was chosen as an a-adrenergic agent which lation of the cat submandibular gland by would be stable under the conditions of autonomic drugs,18 some experiments were the experiments. Clinically it is used in a also made with 2 mM. calcium in the me- 10 per cent solution and has no clear-cut dium. In all cases, drug was present in the effect on eye pressure. It was tested at 0.2 experimental flask only and only during per cent (10~2M) in these experiments. runout. There was not any discernible effect (n = Pilocarpine. Pilocarpine, 10~5 and 10~3 M, 8). was tried without calcium; the latter con- Orciprenaline (Alupent). This was used centration had a highly inhibitory effect as a /?-adrenergic agent instead of isopren- in Berggren's experiment.1 In the present aline; it was hoped to be somewhat more experiment the general shape of the curves stable under our conditions. At 10~5 and was hardly affected except at the begin- 10"2 M it had no discernible effect (n = 6 ning. Something seemed to have happened in each case). This could have been due already in the first 2 minutes since even at to destruction. that time the experimental preparation Butylsympatol (Vasculat). As a certainly had lost significantly more activity to the more stable /?-adrenergic drug, butylsym- medium (p < 0.001 in Fig. 7, A, but patol was tried. In the absence of calcium p < 0.1 in Fig. 7, B). It goes on los- it had no effect at 10"3M (n = 6). In the ing faster for some little time. If the presence of 2 mM. calcium, 10~3M caused 10 minute values are considered, at which a slight acceleration of runout not limited time the rapid phase should be main- to the initial phase, Fig. 7, C. An attempt ly finished, the same p values are ob- to increase this effect by using 10~'-M tained. If calcium is used in the loading failed; at this concentration virtually no as well as runout medium, the experimental effect was seen. curve with 10~r'M pilocarpine runs 5 per cent below the control, but the shape is Discussion quite the same, and there is no acceleration Embryologically, the two layers of epi- of the initial phase (n = 6). thelial cells of the ciliary processes are ar-

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ranged face to face. The free surface of tribution from the stroma in the present the nonpigmented epithelium is its basal experiments? A few experiments with 22Na, pole. It is not known if the 2 layers of cells done for another purpose but conducted always tend to transport material in the under exactly the same conditions as those same direction, aqueous to blood or blood with rubidium, showed that the sodium to aqueous, or if they sometimes oppose space of the preparation was only about 5 each other. Their histochemistry differs to 10 per cent of the rubidium space. Since markedly,19' 20 and they could have quite sodium fills the stroma, if nothing else, only different pharmacology. The cells may have a small part of the rubidium could have different thresholds for drug actions, caus- been in the stroma at the start of the run- ing a dome-shaped dose-response curve or out. Evidently the conditions during load- one with a step. This could explain the re- ing allowed the epithelium to secrete and sults of Walinder3 in which low doses of to deplete the stroma of fluid.17

pilocarpine caused stimulation, and higher Lack of glucose and O2 together reduced doses caused inhibition of the organic uptake of rubidium to about 40 per cent. anion transport system. Moreover, there Since secretion and shrinkage would have are regional differences between the cells been inhibited under these conditions, the covering different parts of the ciliary pro- volume of the stroma should have been cesses and also different kinds of ciliary larger than usual. Since the rubidium 1 1 15> 10 processes. ' ' All this complicates the amount was distinctly smaller, this also interpretation of data obtained from the shows that rubidium must have been whole tissue and from any single tech- mainly located in the epithelium. nique. We have, therefore, arranged our The relative contributions of the pig- experiments so as to make them compa- 1 1(!> 17 mented and nonpigmented layers in the rable with those of Berggren. ' present experiments are not clear. It is In our experiments the runout of Rb possible that the 2 cell layers (and the could be due to a gradual loss of viability, stroma) contribute differently to the early or it could be due directly or indirectly to and later phases of the runout. normal turnover of electrolytes in the cells. Berggren16 found an almost complete in- Very probably both phenomena contribute, hibition of shrinkage when lithium was especially during the later phases. This is substituted for sodium. In the present ex- unavoidable if one wants to study move- periments the same solution caused a ment of material into the aqueous humor. marked acceleration of the rubidium loss The effects of temperature are unfortu- during the first 10 to 15 minutes. It is nately compatible with both interpreta- tempting to speculate that the potassium tions. Decay in viability should be accel- loss which the rubidium loss indicates was erated by lack of O2, glucose, or both, espe- the reason for the impaired function seen cially during the later phases. Since there by Berggren. was very little such effect it seems probable We have no explanation for the calcium that decay of viability was not the domi- effect, a slowing of the runout observed in nant factor. The fact that the metabolic the present experiments. Berggren has no upset caused by the combined effects of comparable experiments. oxygen lack and glucose lack throughout Pilocarpine. In the experiments without the experiment had no clear-cut effect calcium in the medium, the later course made it seem unnecessary to study the ef- of the runout seems to be identical with or fects of anoxia or substrate depletion sep- without pilocarpine (10~3, 10~5 M). During arately for uptake and runout. the first 5 to 10 minutes, however, the loss In Berggren's experiments fluid trans- of rubidium was faster from the pilocar- port from the stroma through both epithe- pine-treated tissue. lial layers was studied. What was the con- The submaxillary gland of the dog and

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the parotid gland of the sheep stimulated seen. Phenylephrine was selected as the through their nerves lose potassium explo- a-adrenergic stimulant because it is stable. sively to the blood and to the saliva during We have found no references to glandular the first minutes.8' ° This is due to loss of actions of this compound. The closely re- cellular potassium. After the initial loss a lated synephrine (Sympatol) which has new steady state is established. Is the ini- the hydroxyl in the 4-position stimulates tial loss in our experiments a similar phe- the submaxillary gland of the cat.24 Again nomenon? In the perfused submandibular the intermittently acting salivary gland gland of the cat, calcium is essential for cells may not be comparable with our cells. the secretory response to acetylcholine as We have found no data on the action of well as to noradrenaline.ls However, lack phenylephrine or similar compounds on of calcium does not prevent all reactions of tubular cells. There are some experiments this gland to acetylcholine; the secretory with dopamine in the chicken25 and dog2G potentials persist.21 In our pilocarpine ex- in which some stimulation of sodium, chlo- periments with calcium the initial loss of ride, and water excretion was seen. How- rubidium was less than in those without ever, a vascular action of the drug cannot calcium which is the opposite to the situa- be excluded. Thus, we know of no con- tion in the salivary glands. These, however, tinuously secreting gland cell carrying a- are quiescent except when stimulated, while receptors. in the ciliary processes none of the auto- Orciprenaline. Orciprenaline, a /?-adre- nomic receptor blockers stops secretion. nergic drug, had no effect, but since we One should, therefore, be wary of drawing did not check its continued presence in the parallels between the 2 types of glands. medium, the negative result does not set- Now, certainly the kidney tubules must be tle the matter. On the other hand, since continuously active and muscarinic agents it is only some 10 to 40 times less active do have an inhibitory effect on sodium reab- than isoprenaline,27 our doses may have 22 23 sorption in the dog and the chicken. We been too high. believe that this effect is probably more Butylsympatol. With only one phenolic closely related to the one we have ob- group, butylsympatol certainly should be served, and we doubt that the initial loss more stable than orciprenaline. Its calori- of rubidium found by us corresponds to genic and positive chronotropic action in the true excitation loss in previously quies- the dog is some 20 to 30 times weaker than cent salivary gland cells. that of orciprenaline.2S A slight stimulation We have seen virtually no effect of pilo- of runout by 1O~3M was observed in the carpine on the later phase of the runout. presence of calcium; maybe this dose was This could be due to lack of a steady-state too high. effect on the cells. But it is also possible /?-adrenergic receptors are certainly pres- that, once the initial effect is over, the ent in salivary glands, although there are rate-limiting factor for the loss of rubidium considerable species differences.29 The in our experiments is not the state of the presence of /3-receptors in the tubules of epithelial cell proper but the supply of the rat kidney has been inferred from the water and electrolytes through the stroma. antidiuretic effect of very small amounts of In the experiments of Walinder3 with pilo- isoprenaline, which do not affect glomeru- carpine inhibition of the organic anion lar filtration rate or effective renal plasma transport system, late effects must have flow and are antagonized by a specific (3- been present. In the shrinkage experiments blocker.30 of Berggren, a late effect may be present It is interesting to note that Leyd- but not convincingly so (Fig. 2 of refer- hecker,31 at one time, used subconjunctival ence 1). injection of 50 mg. of butylsympatol as a Phenylephrine. No effect whatsoever was provocative test for glaucoma. Maybe part

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of the effect in the human eye could in across the surface membrane of cardiac fact have been stimulation of secretion of purkinje fibres, J. Physiol. 177: 453, 1965. aqueous humor. 14. Kozart, D. M.: Light and electron micro- scopic study of regional morphologic differ- Summing up, we believe we have dem- ences in the processes of the ciliary body in onstrated moderate effects of some auto- the rabbit, INVEST. OPHTH. 7: 15, 1968. nomic drugs on electrolyte economy in iso- 15. Wegner, K.: Regional differences in ultra- lated ciliary processes. It is, however, too structure of the rabbit ciliary processes: The early to correlate these effects with possible effect of anesthetics and fixation procedures, INVEST. OPHTH. 6: 177, 1967. effects on secretion in vivo. 16. Berggren, L.: Effect of composition of medium and of metabolic inhibitors on secretion We wish to thank Mrs. Malin Svensson for in vitro by the ciliary processes of the rabbit technical assistance. eye, INVEST. OPHTH. 4: 83, 1965. 17. Berggren, L.: Direct observation of secretory REFERENCES pumping in vitro of the rabbit eye ciliary 1. Berggren, L.: Effect of parasympathomimetic processes: Influence of ion milieu and carbon- and sympathomimetic drugs on secretion in ic anhydrase influence, INVEST. OPHTH. 3: vitro by the ciliary processes of the rabbit 266, 1964. eye, INVEST. OPHTH. 4: 91, 1965. 18. Douglas, W. W., and Poisner, A. M.: The 2. Becker, B.: The transport of organic anions influence of calcium on the secretory response by the rabbit eye, Am. J. Ophth. 50: 862, of the submaxillary gland to acetylcholine or 1960. to noradrenaline, J. Physiol. 165: 528, 1963. 3. Walinder, P.-E.: Influence of pilocarpine on 19. Cole, D. F.: Aqueous humour formation, iodopyracet and iodide accumulation by rab- Docum. Ophth. 21: 116, 1966. bit ciliary body-iris preparations, INVEST. 20. Hansson, H. P. J.: Histochemical demonstra- OPHTH. 5: 378, 1966. tion of carbonic anhydrase activity in some 4. Walinder, P.-E.: The accumulation of alpha epithelia noted for active transport, Acta aminoisobutyric acid by rabbit ciliary body- physiol. scandinav. 73: 427, 1968. iris preparations, INVEST. OPHTH. 7: 67, 1968. 21. Petersen, O. H., Poulsen, J. H., and Thorn, 5. Ehinger, B.: Adrenergic nerves to the eye N. A.: Secretory potentials, secretory rate and and its adnexa in rabbit and guinea-pig, water permeability of the duct system in the Acta univ. kind, II, No. 20, 1964. cat submandibular gland during perfusion 6. Laties, A., and Jacobowitz, D.: A histochemi- with calcium-free Locke's solution, Acta cal study of the adrenergic and cholinergic physiol. scandinav. 71: 203, 1967. innervation of the anterior segment of the 22. Williams, R. L., Pearson, J. E., Jr., and rabbit eye, INVEST. OPHTH. 3: 592, 1964. Carter, M. K.: The saluretic effects of areco- 7. Ehinger, B.: Ocular and orbital vegetative line hydrochloride infused into the left renal nerves, Acta physiol. scandinav. (Suppl. 268) artery of dogs, J. Pharmacol. & Exper. 67: 1, 1966. Therap. 147: 32, 1965. 8. Burgen, A. S. V.: The secretion of potassium 23. May, G. D., and Carter, M. K.: Unilateral in saliva, J. Physiol. 132: 20, 1956. diuresis by infusing arecoline into renal portal 9. Coats, D. A., Denton, D. A., and Wright, system of hens, Am. J. Physiol. 212: 1351, R. D.: The ionic balances and transferences 1967. of the sheep's parotid gland during maximal 24. Emmelin, N., and Muren, A.: Sensitization stimulation, J. Physiol. 144: 108, 1958. of the submaxillary gland to chemical stimuli, 10. Schneyer, L. H., and Schneyer, C. A.: Effects Acta physiol. scandinav. 24: 103, 1951. of pilocarpine on exchange of K42 in slices of submaxillary gland, Proc. Soc. Exper. Biol. & 25. Sanner, E.: Studies on biogenic amines and Med. 116: 813, 1964. reserpine induced block of the diuretic action 11. Burgen, A. S. V., and Spero, L.: The action of hydrochlorothiazide and theophylline in of acetylcholine and other drugs on the efflux the chicken, Acta pharmacol. et toxicol. of potassium and rubidium from smooth mus- (Suppl.) 22: 1, 1965. cle of the guinea-pig intestine, Brit. J. Phar- 26. Meyer, M. B., McNay, J. L., and Goldberg, macol. 34: 99, 1968. L. I.: Effects of dopamine on renal function 12. Kilpatrick, R., Renschler, H. E., Munro, D. and hemodynamics in the dog, J. Pharmacol. S., and Wilson, G. M.: A comparison of the & Exper. Therap. 156: 186, 1967. distribution of 42K and SGRb in rabbit and 27. Engelhardt, A., Hoefke, W., and Wick, H.: man, J. Physiol. 133: 194, 1956. Zur Pharmakologie des Sympathomimeticums 13. Miiller, P.: Potassium and rubidium exchange 1- (3,5 - dihydroxyphenyl) -1- hydroxy-2-isopro-

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pylaminoethane, Arzneimittel-Forsch. 11: 521, Alimentary canal, Vol. II: Secretion, Wash- 1961. ington, 1967, American Physiological Socie- 28. Strubelt, O.: Die kalorigene und positiv ty- chronotrope Wirkung sympathicomimetischer 30. Lees, P., and Lockett, M. F.: A study of the Amine und ihre Beeinflussung durch Blockade /3-adrenergic receptors in rat kidneys, Brit. der adrenergischen /?-Rezeptoren, Arzneimit- J. Pharmacol. 20: 135, 1963. tel-Forsch. 16: 587, 1966. 31. Leydhecker, W.: Eine neue Belastungsprobe 29. Emmelin, N.: Pharmacology of salivary zur Diagnose des Glaukoms, Klin. Monatsbl. glands, in Handbook of physiology, Sec. 6, Augenh. 123: 568, 1953.

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