[1] 64w fold, respectively. Preparations of ab- dominal spinal cord had little or no ef- fect (Table 1).Failure of earlier experiments (2, 4) to demonstrate a hydrosmotic effect may be due to different urophysial prepara- tions or to differences in the test ani- mals. Urinary bladders of Bufo marinus from Florida showed much lower re- sponses to Gillichthys urophyses, com- pared with Bufo marinus from Hawaii.

[1] 169w Urinary bladders were dissected from rainbow trout (Salmo gairdnerii) im- mediately after decapitation and im- mersed in trout-Ringer solution (2). The distal (cloacal) end of the bladder was attached to a suitable extension fitted to a Statham or a Bionix pressure gauge, and the proximal (renal) end was tied off. The internal space in the strain gauge and the bladder was filled with a 1 to 5 dilution of trout-Ringer solution, the bladder was immersed in an organ bath containing 10 ml of trout-Ringer solution at room tempera- ture, and contractions were recorded on chart paper with a Beckman or a 21 MARCH 1969 Bausch and Lomb amplifier-recorder. Aqueous or acetic acid extracts were added to the organ bath, and responses (contractions per minute) were mea- sured for 5 minutes; the bath was then rinsed out twice with the Ringer solu- tion. A good log dose-response rela- tion was obtained when urophysial ex- tracts from the trout or from the mud- sucker (Gillichthys. mirabilis) were used (Fig. 1 ).

[2] 6w Drying of urophyses in acetone be-

[3] 82w 1.6 .' 1.2 E .°0 .8 . 0.41 0 0.004 0.008 0.016 0.032 0.064 0.128 Urophysis per milliliter bath fluid Fig. 1. Bladder-contracting activity in dif- ferent preparations of trout caudal neu- rosecretory system, showing dose-response relationship. A, Urophysis, acetic acid ex- tract; B, urophysis, whole homogenate in distilled water; C, urophysis, Ringer solution extract (supernatant); D, caudal spinal cord, whole homogenate in Ringer of area containing cell-bodies of caudal neurosecretory neurons; no bladder-con- tracting activity is present in abdominal spinal cord.

[4] 12w Teleostean Urophysis: Stimulation of Contractions of Bladder of the Trout Salmo gairdnerii

[5] 76w Abstract. A new activity of the teleost caudal neurosecretory system is de- scribed. Extracts of the urophysis of the mudsucker Gillichthys mirabilis and of the trout Salmo gairdnerii cause rhythmic contractions of the isolated urinary bladder of the trout. The dose-related response provides the basis for a quan- titative bioassay of this urophysial principle. (a) Isolated small intestine of trout. Effects of bladder-contracting doses of acetylcholine (ACh) and an extract of urophysial laboratory standard preparation (Sa).

[6] 22w (b) Isolated rat uterus. Effects of 5-hydroxytryptamine (5-HT), mammalian oxytocin (Po), 4-serine-8-isoleucine oxytocin (ICT), and an extract of urophysial laboratory standard (Sa).

[7] 195w (c) Rat blood pressure. Effects of arginine vasotocin, arginine vasopressin, and urophysial extract (laboratory, S4). Abbreviations are: AVT, arginine vasotocin; AVP, arginine vasopressin; AVP-TH, sodium thioglycollate-treated AVP; S4-TH, sodium thioglycol- late-treated urophysial laboratory standard, S4; NaCl, 0.9 percent NaCl solution. The effect of AVP is abolished by thioglycollate treatment (rupture of disulfide bond); that of the urophysial material is unaffected. fore extraction and the use of 0.25 percent acetic acid for extraction in- stead of water or Ringer solution did not materially change the amount of bladder-contracting activity; acetic acid extraction seemed to increase the yield of activity (Fig. 1). As little as 1/400 of a mudsucker urophysis (equivalent to 0.05 jtg or less of acetone-dried urophysis powder) per milliliter of bath fluid was enough to produce marked and reproducible responses on the iso- lated bladder. To ensure reproducibility of results and to enable a comparison of findings from individual experiments, and also in anticipation of further studies of the bladder-contracting ac- tivity, a laboratory standard preparation of acetone-dried Gillichthys urophyses was obtained. The mean weight of an acetone-dried urophysis, calculated from six batches of the laboratory standard (820 urophyses), was 21.4 + 0.81 jtg.

[8] 22w 1328 After introduction of the laboratory standard, all dosages and experimental results were related to unit weight (,ug) of the standard preparation.

[9] 196w Other biologically active substances which might occur in the urophysis and thus affect the isolated bladder were tested on the trout bladder and on trout and mudsucker small intestine (Fig. 2a). These included the teleost neuro- hypophysial hormones, 5-hydroxytryptamine, histamine, acetylcholine with and without physostigmine or atropine, adrenalin, and noradrenalin. With the exception of the neurohypophysial hor- mones and of adrenalin and noradrena- lin, these substances also produced con- tractions of the bladder. When similar amounts of the substances listed above and of the urophysial extracts were applied to the isolated rat uterus (Fig. 2b) or tested on rat blood pressure (Fig. 2c), it was apparent that the trout bladder-contracting activity of uroph- ysial extracts was associated with a sub- stance whose pharmacological charac- teristics were different from those of 8-arginine oxytocin, 4-serine-8-isoleu- cine oxytocin (ichthyotocin, isotocin), 5-hydroxytryptamine, histamine, acetyl- choline, adrenalin, and noradrenalin. Thus the assay procedure and the lab- oratory standard preparation may be of use not only in the quantitative estima- tion of an active principle present inr the teleost urophysis, but also in biological and chemical identification of this principle which may prove to be a hormone of the caudal neurosecretory system.

[1] 97w However, the most probable reason for failure to observe the hydrosmotic effect may have been the short test periods used in previous studies. When the water loss was determined for each 15 minutes of a 45-minute experimental period, the weight loss for the first 15 minutes was just slightly higher than control values; the weight loss for the second 15 min- utes was much higher than the first; and the last 15 minutes gave values lower than the second. In addition, in this study, bladders were subjected to uro- physial material for 10 minutes before being tested.

[2] 141w Other agents (such as acetylcholine, catecholamines, serotonin, and brady- kinin) possibly present in nervous tis- sue do not stimulate water across the toad bladder (7). Neurohypophysial peptides are, of course, highly active; however, the delayed response to the urophysial preparation indicates some characteristics different from those of the neurohypophysial peptides. Al- though the urophysial hydrosmotic ef- fect is necessarily to be viewed as a pharmacological one (no urophysis or homologous organ is present in amphib- ians or in any other tetrapods), this activity may reflect an important osmo- regulatory influence as yet to be deline- ated in teleosts. At the moment, it appears that this effect is not due to the same factor responsible for teleost blad- der contraction (6). Nevertheless, the utility of the effect in detecting and assaying a biologically active substance from the caudal neurosecretory system is evident.

[3] 15w FLOR LACANILAO Department of Zoology and Cancer Research Genetics Laboratory, University of California, Berkeley 94720

[4] 117w In determining whether urophysial extracts influenced water movement across the isolated fish urinary bladder, comparable to the effect seen on the toad bladder (1), it was noted that the trout bladder showed rhythmic contrac- tions upon exposure to such extracts (or to homogenates of fresh urophysis in Ringer solution). In view of the continued need for a reliable biological assay for any active principle or principles which may occur in the teleost urophysis, this phenomenon was further analyzed. It is now possible to define this kinetic activity with considerable precision and to propose a quantitative method for assay. The new activity of the caudal neurosecretory system is discussed, and the utility of the bioassay procedure is outlined.

[5] 165w for the hypothetical urophysial hormone (3). Most of the information available on urophysial function points to a rela- tion with osmoregulation. However, the pressor activity described earlier (4) and the present bladder-contracting ac- tivity are kinetic effects. At-the same time, of course, an influence on blood pressure or on bladder function could also be ipso facto related to osmoregula- tion, albeit secondarily so. Furthermore, unlike the hydrosmotic effect on the toad bladder (1), the kinetic effect represents a teleost principle acting upon a teleost target organ. In any case, the bioassay described herein opens the way to mean- ingful studies of the pharmacological, physiological, and chemical properties of the hormone or hormones of the caudal neurosecretory system. Initial findings of experiments aimed at chemi- cal identification of the urophysial active principle(s) (S) support the earlier in- dications (4) that more than one active substance may be present in the teleost urophysis. KARL LEDERIS Department of Zoology and Cancer Research Genetics Laboratory, University of California, Berkeley