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Glucose absorption from the urinary bladder of a crab
Comp. Biochem. PhysioL, 1967, Vol. 20, pp. 313 to 317. Pergamon Press Ltd. Printed in Great Britain
SHORT COMMUNICATION GLUCOSE ABSORPTION FROM THE URINARY BLADDER OF A CRAB WARREN J. GROSS Department of Life Sciences, University of California, Riverside, California, U.S.A.
(Received 25 July 1966) A b s t r a c t - - 1 . Glucose introduced with isosmotic perfusion fluid into the
urinary bladder of the crab Pachygrapsus crassipes disappears from the bladder fluid. This cannot be attributed to bladder evacuation. 2. When glucose and phlorizin in perfusion fluid are similarly introduced, glucose remains in the bladder at about the same concentration as found in the blood. 3. Glucose appears in the bladder fluid of phlorizinized crabs, but is absent from the bladder fluid of untreated animals. 4. It is suggested that the urinary bladder of Pachygrapsus is an important site for glucose reabsorption and therefore is a physiologicallyactive region of the renal system. INTRODUCTION THE processes of urine formation by the antennary glands of the decapod Crustacea are little understood. Although there is considerable evidence that the primary urine in some species is formed by ultrafiltration in much the same manner as it is formed by the glomerular kidney of the vertebrates (Bethe eta/., 1935; Picken, 1935; Riegel & Kirschner, 1950; Kirschner & Wagner, 1955), there is a paucity of information concerning the subsequent processing of the filtrate. The physiology of the antennary gland is reviewed by Lockwood (1952) and Ports & Parry (1964); the morphology of this organ is reviewed by Balss (1944). Now should the primary urine be formed by ultrafiltration, then some mechanism for recapturing essential filtered materials such as glucose would be expected to be present somewhere in the renal organ, and indeed glucose appears in the urine of phlorizinized specimens of Homarus (Burger, 1957), Procambarus, Orconectes and Pacifastacus (Riegel & Kirsehner, 1960), but is absent from the urine of these animals when untreated with phlorizin. This suggests that glucose is filtered with the primary urine but normally is reabsorbed. However, no information is available as to the sites of glucose reabsorption. The present investigation produces evidence that glucose absorption can occur from the urinary bladder of the crab Pachy-
gr apsus crassipes. 313
314
WARRENJ. GRoss
MATERIALS AND METHODS The shore crab Pachygrapsus crassipeswas collected at Laguna, California, and maintained in the laboratory at 15°C in 100% artificial sea water made from the Utility Chemical Company Seven-Seas Marine Mix. Only intermolt crabs larger than 20 g were used. A salinity of 34.3 °/00 was considered to be 100% sea water. Perfusion fluid used to simulate primary urine contained the following concentrations of ions: sodium, 483 mM/1; potassium, 7 mM/1; magnesium, 10 mM/1; calcium, 15 raM/l; chloride, 520 mM/1; and sulfate, 10 mM/l. This approximates the blood ionic and osmotic concentrations of Pachygrapsuswhen immersed in 100°/0 sea water (Gross & Capen, 1966). Osmotic concentrations of fluids were determined to within a 1~/o error by means of a Mechrolab Vapor Pressure Osmometer. Glucose was determined by an adaptation of the enzymatic method of Keston (1956) and Teller (1956) as outlined in the Beckman/Spinco Technical Bulletin No. 6073C. The range of error for this method was about 5 per cent for glucose concentrations of 30 mg per cent. Blood was extracted by puncturing the arthrodial membranes at the bases of the walking legs with a glass pipette. Bladder fluid was removed from or introduced into the nephropore by means of a fine glass cannula. Experiments that follow involved the introduction of isosmotic solutions containing glucose and/or phlorizin into the artificially evacuated bladders of normal and phlorizinized crabs. It was necessary, therefore, to establish that such introduced fluids remained in the bladder during the test period. Gross & Capen (1966) demonstrated that when Pachygrapsus was kept out of the water, test fluids introduced into the bladder were retained for 24 hr or longer. A similar method was employed, therefore, to show that test fluids used in the present investigation also were retained in the bladder for the experimental period. Urine from the bladders of crabs taken from 100% sea water was removed and replaced with isosmotic perfusion fluid which was colored with indigo carmine and contained either (a) 100 nag per cent of phlorizin plus 30 mg per cent of glucose (6 crabs) or (b) only 30 mg per cent of glucose (6 crabs). After the introduction of the colored fluid, the crab was placed in a dry plastic container, the floor of which was covered with white absorbent tissue paper, so if bladder fluid was lost during the period of observation, the white paper would become spotted with the blue dye. Of the 12 crabs tested only one (glucose only) showed a loss of fluid through the nephropore after 6 hr on the white paper, but even this specimen still showed color in the bladder fluid, indicating that only part of the fluid had been ejected. The bladder fluid of all other test specimens was deeply colored after 6 hr, but no spotting of the white paper occurred. It is probable, then, that the test solutions used in this study were held in the bladder for the duration of the following experiments. Urine from a crab that had been immersed in 100% sea water was removed from one of the paired bladders and an approximately equal volume of isosmotic perfusion fluid containing 30 mg per cent glucose and/or 100 mg per cent phlorizin was substituted into the empty bladder. The crab then was kept out of the water
GLUCOSE ABSORPTION FROM URINARY BLADDER OF A CRAB
315
in a dry container for 3 hr after which time the bladder fluid was removed and analyzed for glucose. Crabs receiving phlorizin in the artificial bladder fluid were injected 1 hr before with 0.1 ml of isosmotic perfusion fluid containing 100 mg per cent phlorizin. Thus, phlorizin was present on both sides of the bladder membrane, although not in equal concentrations. Probability values in this investigation were determined by Student's t-test.
RESULTS When glucoseis introduced into the bladder of Pachygrapsusit all but disappears in a period of 3 hr, (Table 1 A), the averageconcentrationbeing less than one-fourth the mean concentration of blood glucose (Table I G). Yet when the animal is phlorizinized more glucose remains in the bladder than when the animal is not phlorizinized (Table 1 B). The difference between means of phlorizinized and unphlorizinized animals is highly significant (P < 0.001). It will be noted, however, that the mean glucose concentration in the bladder fluid of phlorizinized animals (9.76 mg per cent) was considerably less than the 30 mg per cent introduced with the perfusion fluid. Yet, again, it will be observed in Table 1 G that the mean blood glucose concentration of 10 normal animals was 9.45 mg per cent. It is believed therefore that glucose introduced into the bladder, with phlorizin, diffused from a high concentration of 30 mg per cent to the lower concentration in the blood until an equilibrium was attained at the blood glucose concentration. TABLE l--GLucOSE CONCENTRATIONS(mg %) IN FLUIDSOF Pachygrapsus Bladder fluid
A B C D E F
Initial bladder fluid
No.
Mean
S.D.
Glucose (30 mg %) in perfusion fluid Glucose (30 mg %) plus phlorizin (100 mg %) in perfusion fluid Phlorizin (100 mg %) in perfusion fluid Perfusion fluid only Normal urine from full bladder Urine 2 hr after bladder evacuation
21
2"14
1"71
17 16 9 10 6
9.76 4"61 0 0 0
6"69 3"87 0 0 0
10
9"45
3"43
Blood G
Normal crabs from 100% sea water
When phlorizin alone was introduced with perfusion fluid into the bladder of
Pachygrapsus significant amounts of glucose could be recovered from the bladder fluid after 3 hr (Table 1 C). The mean concentration, 4.61 mg per cent, is significantly less than the mean, 9.76, for crabs given both glucose and phlorizin
316
WARRENJ. GRoss
(P < 0.02) and greater than the mean, 2.14, for crabs receiving only glucose in the bladder fluid (P < 0-05). Since the bladder was full and the animal was kept out of the water, it seems unlikely that significant amounts of urine entered the bladder from the labyrinth during the experiment, therefore the probable effect of phlorizin was to permit glucose to diffuse from the blood into the bladder. However, the concentration of glucose observed in animals receiving both glucose and phlorizin is too high to be caused entirely by the movement of glucose into the bladder from the blood. Part, therefore, must be the remainder of that originally introduced into the bladder. When perfusion fluid containing neither glucose nor phlorizin was introduced, no glucose could be detected in the bladder fluid after 3 hr (Table 1 D). Glucose is neither present in the urine of normal animals with full bladders taken from 100% sea water (Table 1 E), nor is it detectable in the urine taken 2 hr after artificial evacuation of the bladder in crabs from 100~o sea water (Table 1 F). Volumes of urine sufficient for glucose analysis could not be collected earlier than 2 hr after bladder evacuation. While no evidence is presented that glucose is contained in the urine as it enters the bladder, data in Table 1 A suggest that such glucose would have been rapidly reabsorbed and probably undetectable. DISCUSSION The evidence presented in Table 1 indicates that glucose can be absorbed from the urinary bladder of the crab Pachygrapsus crassipes. Also there is evidence that phlorizin blocks absorption, and glucose will diffuse from the blood into the bladder fluid of a phlorizinized animal (Table 1 B and C). It is apparent, therefore, that the appearance of glucose in the urine of a phlorizinized crab is doubtful evidence that the primary urine is formed by ultrafiltration under hydrostatic pressure. Although the bladder be capable of glucose absorption, it may not be the principal site for this activity in the renal organ. Glucose may also be reabsorbed in the labyrinth, but to determine this would require techniques of micro-puncture that have not yet been mastered. Clearly, the glucose-transporting function of the bladder would prevent the loss of glucose from the blood into the urine by diffusion. On the other hand, Gross & Capen (1966) have shown that the urinary bladder of Pachygrapsus is physiologically active and, indeed, is important in the regulation of magnesium. The present investigation produces further evidence that the bladder is functionally a significant part of the renal apparatus of crabs.
Acknowledgements--These studies were supported by the National Science Foundation Grant, GB-3969. I wish to acknowledge the able technical assistance of Messrs. John Armstrong, John Bachman and Ronald Capen. REFERENCES
BALSSH. (1944) Decapoda. In Bronn's K1. Ordn. Tierreichs, Bd. 5, Abt. 1, Bch. 7, Lfg. 4, 562-591. BETHEA., HOLSTE. YON &; HUF E. (1935) Die Bedeutung des mechanlschen Innendrucks fiir die Anpassung gepanzerter Seetiere an 2~nderungen des osmotischen Aussendrucks. Pflagers Arch. ges. Physiol. 235, 330-344.
G L U C O S E A B S O R P T I O N FROM U R I N A R Y BLADDER OF A CRAB
317
BURGER J. W. (1957) The general form of excretion in the lobster, Homarus. Biol. Bull., Woods Hole 113, 207-223. GROSS W. J. & CAP~,~r R. (1966) Some functions of the urinary bladder in a crab. Biol. Bull., Woods Hole. (In press.) K~STON A. S. (1956) Specific colorimetric enzymatic analytical reagents for glucose. Abstr. Pap., 129th Meeting, Am. chem. Soc., 31C. KIRSCHNER L. B. & WAGNERS. (1965) The site and permeability of the filtration locus in the crayfish antennal gland, ft. exp. Biol. 43, 385-395. LOCKWOODA. P. M. (1962) The osmoregulation of Crustacea. Biol. Rev. 37, 257-305. PmKEN L. E. R. (1936) The mechanism of urine formation in invertebrates--I. The excretion mechanism in certain Arthropoda. J. exp. Biol. 13, 309-328. POTTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Macmillan, New York. RIEOEL J. A. & KIRSCHNERL. B. (1960) The excretion of inulin and glucose by the crayfish antennal gland. Biol. Bull., Woods Hole 118, 296-307. TELLER J. D. (1956) Direct, quantitative, colorimetric determination of serum or plasma glucose. Abstr. Pap., 130th Meeting, Am. chem. Soc., 69C.
SHORT COMMUNICATION GLUCOSE ABSORPTION FROM THE URINARY BLADDER OF A CRAB WARREN J. GROSS Department of Life Sciences, University of California, Riverside, California, U.S.A.
(Received 25 July 1966) A b s t r a c t - - 1 . Glucose introduced with isosmotic perfusion fluid into the
urinary bladder of the crab Pachygrapsus crassipes disappears from the bladder fluid. This cannot be attributed to bladder evacuation. 2. When glucose and phlorizin in perfusion fluid are similarly introduced, glucose remains in the bladder at about the same concentration as found in the blood. 3. Glucose appears in the bladder fluid of phlorizinized crabs, but is absent from the bladder fluid of untreated animals. 4. It is suggested that the urinary bladder of Pachygrapsus is an important site for glucose reabsorption and therefore is a physiologicallyactive region of the renal system. INTRODUCTION THE processes of urine formation by the antennary glands of the decapod Crustacea are little understood. Although there is considerable evidence that the primary urine in some species is formed by ultrafiltration in much the same manner as it is formed by the glomerular kidney of the vertebrates (Bethe eta/., 1935; Picken, 1935; Riegel & Kirschner, 1950; Kirschner & Wagner, 1955), there is a paucity of information concerning the subsequent processing of the filtrate. The physiology of the antennary gland is reviewed by Lockwood (1952) and Ports & Parry (1964); the morphology of this organ is reviewed by Balss (1944). Now should the primary urine be formed by ultrafiltration, then some mechanism for recapturing essential filtered materials such as glucose would be expected to be present somewhere in the renal organ, and indeed glucose appears in the urine of phlorizinized specimens of Homarus (Burger, 1957), Procambarus, Orconectes and Pacifastacus (Riegel & Kirsehner, 1960), but is absent from the urine of these animals when untreated with phlorizin. This suggests that glucose is filtered with the primary urine but normally is reabsorbed. However, no information is available as to the sites of glucose reabsorption. The present investigation produces evidence that glucose absorption can occur from the urinary bladder of the crab Pachy-
gr apsus crassipes. 313
314
WARRENJ. GRoss
MATERIALS AND METHODS The shore crab Pachygrapsus crassipeswas collected at Laguna, California, and maintained in the laboratory at 15°C in 100% artificial sea water made from the Utility Chemical Company Seven-Seas Marine Mix. Only intermolt crabs larger than 20 g were used. A salinity of 34.3 °/00 was considered to be 100% sea water. Perfusion fluid used to simulate primary urine contained the following concentrations of ions: sodium, 483 mM/1; potassium, 7 mM/1; magnesium, 10 mM/1; calcium, 15 raM/l; chloride, 520 mM/1; and sulfate, 10 mM/l. This approximates the blood ionic and osmotic concentrations of Pachygrapsuswhen immersed in 100°/0 sea water (Gross & Capen, 1966). Osmotic concentrations of fluids were determined to within a 1~/o error by means of a Mechrolab Vapor Pressure Osmometer. Glucose was determined by an adaptation of the enzymatic method of Keston (1956) and Teller (1956) as outlined in the Beckman/Spinco Technical Bulletin No. 6073C. The range of error for this method was about 5 per cent for glucose concentrations of 30 mg per cent. Blood was extracted by puncturing the arthrodial membranes at the bases of the walking legs with a glass pipette. Bladder fluid was removed from or introduced into the nephropore by means of a fine glass cannula. Experiments that follow involved the introduction of isosmotic solutions containing glucose and/or phlorizin into the artificially evacuated bladders of normal and phlorizinized crabs. It was necessary, therefore, to establish that such introduced fluids remained in the bladder during the test period. Gross & Capen (1966) demonstrated that when Pachygrapsus was kept out of the water, test fluids introduced into the bladder were retained for 24 hr or longer. A similar method was employed, therefore, to show that test fluids used in the present investigation also were retained in the bladder for the experimental period. Urine from the bladders of crabs taken from 100% sea water was removed and replaced with isosmotic perfusion fluid which was colored with indigo carmine and contained either (a) 100 nag per cent of phlorizin plus 30 mg per cent of glucose (6 crabs) or (b) only 30 mg per cent of glucose (6 crabs). After the introduction of the colored fluid, the crab was placed in a dry plastic container, the floor of which was covered with white absorbent tissue paper, so if bladder fluid was lost during the period of observation, the white paper would become spotted with the blue dye. Of the 12 crabs tested only one (glucose only) showed a loss of fluid through the nephropore after 6 hr on the white paper, but even this specimen still showed color in the bladder fluid, indicating that only part of the fluid had been ejected. The bladder fluid of all other test specimens was deeply colored after 6 hr, but no spotting of the white paper occurred. It is probable, then, that the test solutions used in this study were held in the bladder for the duration of the following experiments. Urine from a crab that had been immersed in 100% sea water was removed from one of the paired bladders and an approximately equal volume of isosmotic perfusion fluid containing 30 mg per cent glucose and/or 100 mg per cent phlorizin was substituted into the empty bladder. The crab then was kept out of the water
GLUCOSE ABSORPTION FROM URINARY BLADDER OF A CRAB
315
in a dry container for 3 hr after which time the bladder fluid was removed and analyzed for glucose. Crabs receiving phlorizin in the artificial bladder fluid were injected 1 hr before with 0.1 ml of isosmotic perfusion fluid containing 100 mg per cent phlorizin. Thus, phlorizin was present on both sides of the bladder membrane, although not in equal concentrations. Probability values in this investigation were determined by Student's t-test.
RESULTS When glucoseis introduced into the bladder of Pachygrapsusit all but disappears in a period of 3 hr, (Table 1 A), the averageconcentrationbeing less than one-fourth the mean concentration of blood glucose (Table I G). Yet when the animal is phlorizinized more glucose remains in the bladder than when the animal is not phlorizinized (Table 1 B). The difference between means of phlorizinized and unphlorizinized animals is highly significant (P < 0.001). It will be noted, however, that the mean glucose concentration in the bladder fluid of phlorizinized animals (9.76 mg per cent) was considerably less than the 30 mg per cent introduced with the perfusion fluid. Yet, again, it will be observed in Table 1 G that the mean blood glucose concentration of 10 normal animals was 9.45 mg per cent. It is believed therefore that glucose introduced into the bladder, with phlorizin, diffused from a high concentration of 30 mg per cent to the lower concentration in the blood until an equilibrium was attained at the blood glucose concentration. TABLE l--GLucOSE CONCENTRATIONS(mg %) IN FLUIDSOF Pachygrapsus Bladder fluid
A B C D E F
Initial bladder fluid
No.
Mean
S.D.
Glucose (30 mg %) in perfusion fluid Glucose (30 mg %) plus phlorizin (100 mg %) in perfusion fluid Phlorizin (100 mg %) in perfusion fluid Perfusion fluid only Normal urine from full bladder Urine 2 hr after bladder evacuation
21
2"14
1"71
17 16 9 10 6
9.76 4"61 0 0 0
6"69 3"87 0 0 0
10
9"45
3"43
Blood G
Normal crabs from 100% sea water
When phlorizin alone was introduced with perfusion fluid into the bladder of
Pachygrapsus significant amounts of glucose could be recovered from the bladder fluid after 3 hr (Table 1 C). The mean concentration, 4.61 mg per cent, is significantly less than the mean, 9.76, for crabs given both glucose and phlorizin
316
WARRENJ. GRoss
(P < 0.02) and greater than the mean, 2.14, for crabs receiving only glucose in the bladder fluid (P < 0-05). Since the bladder was full and the animal was kept out of the water, it seems unlikely that significant amounts of urine entered the bladder from the labyrinth during the experiment, therefore the probable effect of phlorizin was to permit glucose to diffuse from the blood into the bladder. However, the concentration of glucose observed in animals receiving both glucose and phlorizin is too high to be caused entirely by the movement of glucose into the bladder from the blood. Part, therefore, must be the remainder of that originally introduced into the bladder. When perfusion fluid containing neither glucose nor phlorizin was introduced, no glucose could be detected in the bladder fluid after 3 hr (Table 1 D). Glucose is neither present in the urine of normal animals with full bladders taken from 100% sea water (Table 1 E), nor is it detectable in the urine taken 2 hr after artificial evacuation of the bladder in crabs from 100~o sea water (Table 1 F). Volumes of urine sufficient for glucose analysis could not be collected earlier than 2 hr after bladder evacuation. While no evidence is presented that glucose is contained in the urine as it enters the bladder, data in Table 1 A suggest that such glucose would have been rapidly reabsorbed and probably undetectable. DISCUSSION The evidence presented in Table 1 indicates that glucose can be absorbed from the urinary bladder of the crab Pachygrapsus crassipes. Also there is evidence that phlorizin blocks absorption, and glucose will diffuse from the blood into the bladder fluid of a phlorizinized animal (Table 1 B and C). It is apparent, therefore, that the appearance of glucose in the urine of a phlorizinized crab is doubtful evidence that the primary urine is formed by ultrafiltration under hydrostatic pressure. Although the bladder be capable of glucose absorption, it may not be the principal site for this activity in the renal organ. Glucose may also be reabsorbed in the labyrinth, but to determine this would require techniques of micro-puncture that have not yet been mastered. Clearly, the glucose-transporting function of the bladder would prevent the loss of glucose from the blood into the urine by diffusion. On the other hand, Gross & Capen (1966) have shown that the urinary bladder of Pachygrapsus is physiologically active and, indeed, is important in the regulation of magnesium. The present investigation produces further evidence that the bladder is functionally a significant part of the renal apparatus of crabs.
Acknowledgements--These studies were supported by the National Science Foundation Grant, GB-3969. I wish to acknowledge the able technical assistance of Messrs. John Armstrong, John Bachman and Ronald Capen. REFERENCES
BALSSH. (1944) Decapoda. In Bronn's K1. Ordn. Tierreichs, Bd. 5, Abt. 1, Bch. 7, Lfg. 4, 562-591. BETHEA., HOLSTE. YON &; HUF E. (1935) Die Bedeutung des mechanlschen Innendrucks fiir die Anpassung gepanzerter Seetiere an 2~nderungen des osmotischen Aussendrucks. Pflagers Arch. ges. Physiol. 235, 330-344.
G L U C O S E A B S O R P T I O N FROM U R I N A R Y BLADDER OF A CRAB
317
BURGER J. W. (1957) The general form of excretion in the lobster, Homarus. Biol. Bull., Woods Hole 113, 207-223. GROSS W. J. & CAP~,~r R. (1966) Some functions of the urinary bladder in a crab. Biol. Bull., Woods Hole. (In press.) K~STON A. S. (1956) Specific colorimetric enzymatic analytical reagents for glucose. Abstr. Pap., 129th Meeting, Am. chem. Soc., 31C. KIRSCHNER L. B. & WAGNERS. (1965) The site and permeability of the filtration locus in the crayfish antennal gland, ft. exp. Biol. 43, 385-395. LOCKWOODA. P. M. (1962) The osmoregulation of Crustacea. Biol. Rev. 37, 257-305. PmKEN L. E. R. (1936) The mechanism of urine formation in invertebrates--I. The excretion mechanism in certain Arthropoda. J. exp. Biol. 13, 309-328. POTTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Macmillan, New York. RIEOEL J. A. & KIRSCHNERL. B. (1960) The excretion of inulin and glucose by the crayfish antennal gland. Biol. Bull., Woods Hole 118, 296-307. TELLER J. D. (1956) Direct, quantitative, colorimetric determination of serum or plasma glucose. Abstr. Pap., 130th Meeting, Am. chem. Soc., 69C.