Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (non-lens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 3.5-4.0. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.     

THE POTATO TUBER NEMATODE, DITYLENCHUS DESTRUCTOR THORNE, 1945; THE CAUSE OF EELWORM DISEASE IN BULBOUS IRIS

For about twenty-five years a fairly widespread disease of bulbous iris has been known, the cause of which has always been attributed to a biologic race of the stem eelworm Ditylenchus dipsaci (Kühn, 1857), Filipjev, 1936.
Thorne (1945) showed that the nematode causing rot in potato tubers is different from D. dipsaci and named it D. destructor. This nematode has since been reported from Mentha arvensis L. by Hurst (1948) and from Sonchus arvensis L. by Goodey & Goodey (1949).
Examination of nematodes from diseased iris bulbs showed them to possess rounded tail tips and six incisures on each lateral field; characters by which Ditylenchus destructor is distinguished from D. dipsaci.
Cross-inoculation experiments showed that the eelworm causing disease in potato tubers would invade and set up characteristic symptoms in iris bulbs and, in the opposite direction, the eelworm responsible for disease in iris bulbs would give rise to characteristic symptoms in potato tubers. Transfer was also effected from potato and iris to Mentha arvensis and from iris to Sonchus arvensis.
The history of the disease in bulbous iris is briefly reviewed and the biology of Ditylenchus destructor discussed and compared with that of D. dipsaci.
The conclusion that D. destructor is the nematode causing eelworm disease of bulbous iris has been reported earlier (Goodey, J. B. 1950).     

The effects of alpha 1-adrenergic and muscarinic cholinergic stimulation on prostaglandin release by rabbit iris

We have investigated the effects of norepinephrine (NE) and acetylcholine (ACh) on prostaglandin (PGE2 and 6 keto-PGF1 alpha) production by rabbit iris, measured by radioimmunoassay (RIA), and the type of phospholipase activated by NE in irides in which phosphatidylinositol (PI) was doubly prelabeled with [3H] myo-inositol and [1-14C] arachidonic acid (14C-AA), quantitated by radiometric and chromatographic methods. PGE2 output in 60 min (3.6 micrograms/g tissue) was 2.6 times greater than 6 keto-PGF1 alpha. PG production is time-dependent and it is stimulated by NE and ACh in a dose-dependent manner. The NE- and ACh-induced release of PGE2, measured by RIA, is mediated through alpha 1-adrenergic and muscarinic cholinergic receptors, respectively, and it requires Ca2+ for maximal stimulation. Studies on the mechanism of AA release from PI in irides doubly prelabeled with 14C-AA and [3H] myo-inositol revealed the following: (a) Both NE and ACh increased the breakdown of PI, and this was accompanied by a significant increase in the release of AA and consequently PGE2. The stimulatory effects of NE and ACh are mediated through alpha 1-adrenergic and muscarinic cholinergic receptors respectively. (b) The NE-induced formation of 3H-lyso PI and the NE-induced metabolism of 14C-1,2-diacyl-glycerol (DG) are time-dependent. Two pathways for AA release from PI are probably operative in the iris: (a) An indirect release by PI-specific phospholipase C which produces DG, followed by the actions of DG- and monoacylglycerol lipases on DG to release AA. (b) A direct release by phospholipase A2. Whether lyso PI is a product of the polyphosphoinositide response remains to be established. Other phospholipids such as phosphatidylcholine and phosphatidylethanolamine could also serve as a source for AA in PG synthesis. In conclusion, the data presented provide evidence that in the iris the neuro-transmitter-stimulated release of PG and AA, from phosphoinositides, for PG synthesis is coupled to the activation of alpha 1-adrenergic and muscarinic cholinergic receptors.     

Technical note: quantitative measures of iris color using high resolution photographs

Our understanding of the genetic architecture of iris color is still limited. This is partly related to difficulties associated with obtaining quantitative measurements of eye color. Here we introduce a new automated method for measuring iris color using high resolution photographs. This method extracts color measurements in the CIE 1976 L*a*b* (CIELAB) color space from a 256 by 256 pixel square sampled from the 9:00 meridian of the iris. Color is defined across three dimensions: L* (the lightness coordinate), a* (the red-green coordinate), and b* (the blue-yellow coordinate). We applied this method to a sample of individuals of diverse ancestry (East Asian, European and South Asian) that was genotyped for the HERC2 rs12913832 polymorphism, which is strongly associated with blue eye color. We identified substantial variation in the CIELAB color space, not only in the European sample, but also in the East Asian and South Asian samples. As expected, rs12913832 was significantly associated with quantitative iris color measurements in subjects of European ancestry. However, this SNP was also strongly associated with iris color in the South Asian sample, although there were no participants with blue irides in this sample. The usefulness of this method is not restricted only to the study of iris pigmentation. High-resolution pictures of the iris will also make it possible to study the genetic variation involved in iris textural patterns, which show substantial heritability in human populations.     

Potentiation of sympathetic neurotransmission in bovine isolated irides by isoprostanes.

Isoprostanes (IsoP) are formed by free radical catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. In the present study, we examined the effect of IsoP on norepinephrine (NE) release from the bovine isolated iris. Furthermore, we studied the role of IsoP's in hydrogen peroxide (H2O2)-induced enhancement of NE release from this tissue. Isolated bovine irides were prepared for studies of [3H]NE release using the superfusion method. Release of [3H]NE was induced via electrical field stimulation. Both 8-iso-prostaglandin E2 (E2-IsoP) and 8-iso-prostaglandin F2 alpha (F2-IsoP) produced a concentration-related enhancement of field-stimulated [3H]NE release from isolated bovine irides, an effect that was mimicked by the thromboxane (Tx) receptor agonist, U46619 and by H2O2. The Tx-receptor antagonist, SQ 29548 inhibited responses to E2-IsoP (10 microM) with an IC50 of 370 +/- 50 nM. SQ 29548 (10 microM) also blocked the enhancement of electrically-evoked [3H]NE release induced by U46619 (10 microM) but not that caused by H2O2 (300 microM). The Tx synthetase inhibitor, carboxyheptylimidazole (10 microM) prevented the stimulatory effect of E2-IsoP on evoked [3H]NE release without affecting responses induced by H2O2. We conclude that IsoP's can enhance sympathetic neurotransmission in the bovine isolated iris, an effect that can be blocked by a Tx-receptor antagonist. Furthermore, endogenously produced Tx's mediate the stimulatory effect of IsoP's on NE release. However, endogenously generated IsoP's or Tx's are not involved in H2O2-induced potentiation of sympathetic neurotransmission.     

A scanning electron microscopic and quantitative histologic description of lens regeneration in the newt,Notophthalmus viridescens

Adult newts (Notophthalmus viridescens) were lentectomized and at intervals from 4 to 21 days after lentectomy iridocorneal complexes from these animals were examined by scanning electron microscopy to allow a full appreciation for the shape of the regenerating lens. Until around day 12 after lentectomy the posterior surface of the iris is covered by a dense mat of fibrous material which cannot be removed without damage to the iris and which obscures the events of cytoplasmic shedding. The regenerate becomes visible first around stage IV (day 12). A small but clear groove demarcates the regenerate from the rest of the iris. As regeneration progresses there is a marked reduction in debris on the iris surface and the regenerate appears as a U-shaped thickening occupying about one-third of the dorsal half of the iris. During later stages (VI–X) the regenerate protrudes into the pupil inferiorly and posteriorly towards the retina, but does not encroach laterally on the remaining pigmented iris tissue. Prior to secretion of the lens capsule the outline of individual cells is visible on the surface of the regenerate and some regenerates exhibit a prominent dimple on their posterior aspects. Following secretion of the capsule the surface of the regenerate becomes smooth. Quantitative studies show that volume and maximum section area of the regenerate are both more strongly correlated with developmental stage of regeneration than with time after lentectomy.     

Increased iris-lens contact following spontaneous blinking: Mathematical modeling

The purpose of this work was to study in silico how iris root rotation due to spontaneous blinking alters the iris contour. An axisymmetric finite-element model of the anterior segment was developed that included changes in the iris contour and the aqueous humor flow. The model geometry was based on average values of ocular dimensions. Blinking was modeled by rotating the iris root posteriorly and returning it back to the anterior. Simulations with maximum rotations of 2°, 4°, 6°, and 8° were performed. The iris-lens contact distance and the pressure difference between the posterior and anterior chambers were calculated. When the peak iris root rotation was 2°, the maximum iris-lens contact increased gradually from 0.28 to 0.34mm within eight blinks. When the iris root was rotated by 6° and 8°, the pressure difference between the posterior and anterior chambers dropped from a positive value (1.23Pa) to negative values (-0.86 and -1.93Pa) indicating the presence of reverse pupillary block. Apparent iris-lens contact increased with steady blinking, and the increase became more pronounced as posterior rotation increased. We conclude that repeated iris root rotation caused by blinking could maintain the iris in a posterior position under normal circumstances, which would then lead to the clinically observed anterior drift of the iris when blinking is prevented.     

Transdifferentiation of Adult Frog Iris in Retina or Lens by Exogeneous Influences

The mechanisms of transdifferentiation of iris epithelial cells of Rana temporaria (Anura) in culture depending on influences from different sources were studied. In terminally differentiated iris cells, the process of transdifferentiation is initiated by dedifferentiation. Melanosomes are shed from iris cells due to cell surface activity. After depigmentation, iris epithelial cells become capable of proliferating and competent to react to the influences of various exogenous factors. Under the influence of retinal factors secreted by lentectomized tadpole eyes, both dorsal and ventral irises are converted to neural retina. Under the influence of factors from eye vesicles, the irises are converted to neural retina as well. Similar results were obtained in transfilter experiments, in which a 3-day period of transfilter interaction between the irises and eye vesicles ensured depigmentation of the iris followed by transdifferentiation into complete NR with visual receptor. Lentoid formation occurred under the influence of adult frog lens epithelium. Immunofluorescent analysis confirmed the lens nature of the lentoids. In control experiments under the conditions of the tadpole eye orbit, in which programming influences were absent, iris epithelial cells remained unaffected.
The problem of true cell-reprogramming to new differentiation in contrast to expression of inherent properties of the iris epithelial cells is discussed.     

Three genome-wide association studies and a linkage analysis identify HERC2 as a human iris color gene

Human iris color was one of the first traits for which Mendelian segregation was established. To date, the genetics of iris color is still not fully understood and is of interest, particularly in view of forensic applications. In three independent genome-wide association (GWA) studies of a total of 1406 persons and a genome-wide linkage study of 1292 relatives, all from the Netherlands, we found that the 15q13.1 region is the predominant region involved in human iris color. There were no other regions showing consistent genome-wide evidence for association and linkage to iris color. Single nucleotide polymorphisms (SNPs) in the HERC2 gene and, to a lesser extent, in the neighboring OCA2 gene were independently associated to iris color variation. OCA2 has been implicated in iris color previously. A replication study within two populations confirmed that the HERC2 gene is a new and significant determinant of human iris color variation, in addition to OCA2. Furthermore, HERC2 rs916977 showed a clinal allele distribution across 23 European populations, which was significantly correlated to iris color variation. We suggest that genetic variants regulating expression of the OCA2 gene exist in the HERC2 gene or, alternatively, within the 11.7 kb of sequence between OCA2 and HERC2, and that most iris color variation in Europeans is explained by those two genes. Testing markers in the HERC2-OCA2 region may be useful in forensic applications to predict eye color phenotypes of unknown persons of European genetic origin.     

Anoxic stress leads to hydrogen peroxide formation in plant cells.

Hydrogen peroxide (H2O2) was detected cytochemically in plant tissues during anoxia and re-oxygenation by transmission electron microscopy using its reaction with cerium chloride to produce electron dense precipitates of cerium perhydroxides. Anoxia-tolerant yellow flag iris (Iris pseudacorus) and rice (Oryza sativa), and anoxia-intolerant wheat (Triticum aestivum) and garden iris (Iris germanica) were used in the experiments. In all plants tested, anoxia and re-oxygenation increased H2O2 in plasma membranes and the apoplast. In the anoxia-tolerant species the response was delayed in time, and in highly tolerant I. pseudacorus plasma membrane associated H2O2 was detected only after 45 d of oxygen deprivation. Quantification of cerium precipitates showed a statistically significant increase in the amount of H2O2 caused by anoxia in wheat root meristematic tissue, but not in the anoxia-tolerant I. pseudacorus rhizome parenchyma. Formation of H2O2 under anoxia is considered mainly an enzymatic process (confirmed by an enzyme inhibition analysis) and is due to the trace amount of dissolved oxygen (below 10(-5) M) present in the experimental system. The data suggest oxidative stress is an integral part of oxygen deprivation stress, and emphasize the importance of the apoplast and plasma membrane in the development of the anoxic stress response.     

Fine structure of the photosensitive iris of the toad, Bufo marinus

The iris of the toad Bufo marinus is directly photosensitive and will constrict in response to light striking only the iris. This is true even when the iris is isolated from the rest of the eye, and therefore from reflex neuronal influences initiated in the retina. This autonomous response is probably mediated by the sphincter pupillae muscle, since no specialized photoreceptors are present in the iris, nor does the sphincter exhibit any specializations likely to subserve a purely photoreceptive function. The photosensitive sphincter appears typical of smooth muscle and, like mammalian sphincters, possesses many intercellular junctions. The iris possesses a well-developed neuronal plexus with fibers projecting into the sphincter muscle layer. Nerve terminals contain small, agranular (30-70nm) and large, dense-cored (80-120nm) vesicles. No consistent postsynaptic specializations are seen on any cells of the iris, including the cells of the sphincter muscle. The anterior pigment epithelial cells of the iris appear specialized and resemble the myoepithelial dilator muscle described by Kelly and Arnold ('72) for the iris of rats.     

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [³H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.     

Enhancement of RNA labeling in iris and lens by wounding cornea

Lentectomy of the newt eye leads to formation of the lens from the iris. The initial event which occurs in the iris after lentectomy is enhancement of uridine incorporation into RNA. The present data demonstrate that surgery on the cornea without lentectomy enhances uridine incorporation into iris RNA. However, the profile of incorporation after cornea surgery is different from that after lentectomy. Furthermore, cornea surgery fails to cause the high level of incorporation of thymidine into iris which occurs after lentectomy. Cornea surgery also causes enhancement of uridine incorporation into lens RNA with a profile different from that in iris RNA.     

Monogenic control of iris coloration in the January tetra (Hemigrammus hyanuary Characidae)

The January tetra (Hemigrammus hyanuary Durbin) exhibits two eye color phenotypes. These have a silver iris, which is characteristic of the species, and a green color variant. Segregation patterns observed in the progenies from 12 different crosses support an hypothesis for the monogenic inheritance of iris coloration in this species.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.     

Transformation of Iris into Lens in vitro and its Dependency on Neural Retina

Upon lentectomy of adult newt eyes, the dorsal iris epithelium produces a cell population that develops into a new lens. The tissue transformation can be completed not only in the isolated lentectomized eye cultured as a whole, but also in the isolated newt normal dorsal iris combined with the retina of frog larvae in vitro. In this study, 93% of such cultures produced lens tissue made up of newt cells. Well-differentiated lens fibre cells were formed which showed positive immunofluorescence for gamma crystallins. When the isolated dorsal iris epithelium was cultured under the same conditions, well-differentiated lens tissue was again formed in 95% of the cases, suggesting that iris epithelial cells and not iris stromal cells are responsible for lens formation. In contrast, the combination of newt ventral iris with frog retina did not produce any newt lens tissue. No lens tissue was produced when the dorsal iris was cultured with newt spleen or lung, although a considerable number of iris epithelial cells became depigmented. Isolated normal dorsal iris or normal dorsal iris epithelium cultured alone infrequently produced a population of depigmented cells but failed to form lens tissue. On the basis of the present and earlier data, it is concluded that in Wolffian lens regeneration in situ , interaction of the iris epithelial cells with the retina plays a decisive role. However, it is suggested that the iris epithelial cells may have an inherent tendency towards lens formation, and that the factor(s) from the retina facilitates the realization of this tendency, rather than instructing the cells to produce lens. The reported experiments provide the first direct evidence for the existence of cellular metaplasia by demonstrating transformation of fully differentiated iris epithelial cells into lens cells.     

In Vivo Electrical Stimulation of the Sympathetic Nerve of the Eye Increases Inositol Phosphate Production and Prostaglandin Release in the Rabbit Iris Muscle

The effects of in vivo electrical stimulation of the sympathetic nerve of the eye on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in rabbit iris and release of arachidonate and prostaglandin (PG) E2 into aqueous humor were investigated. myo-[3H]Inositol or [1-14C]arachidonate was injected intracamerally into each eye 3 h before electrical stimulation of one of the sympathetic trunks. Tissue phosphoinositides were determined by TLC, and 3H-labeled inositol phosphates were analyzed by either ion-exchange chromatography or HPLC. The aqueous humor was analyzed for 14C-labeled arachidonate and PGE2 by radiochromatography and for unlabeled PGE2 by radioimmunoassay. The results obtained from this study can be summarized as follows: (a) The rates of in vivo incorporation of myo-[3H]inositol into phosphoinositides and accumulation of 3H-labeled inositol phosphates in the iris muscle increased with time and then leveled off between 3 and 5 h. (b) Distribution of 3H radioactivity in inositol phosphates, as determined by HPLC, showed that of the total radioactivity in inositol phosphates, 53.6% was recovered in myo-inositol 1-phosphate, 36% in myo-inositol bisphosphate, 0.95% in myo-inositol 1,3,4-trisphosphate (1,3,4-IP3), and 2.6% in 1,4,5-IP3. (c) Electrical stimulation of the sympathetic nerve resulted in a significant loss of 3H radioactivity from PIP2 and a concomitant increase of that in IP3, an observation indicating that PIP2 is the physiological substrate for alpha 1-adrenergic receptors in this tissue. (d) Release of IP3 and liberation of arachidonate for PGE2 synthesis are dependent on the duration of stimulation and the intensity (voltage) of stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)