Neurochemical Research, Vol. 18, No. 5, 1993, pp. 639-645 Radio-Label and Mass Determinations of Inositol 1,3,4,5- Tetrakisphosphate Formation in Rat Cerebral Cortical Slices: Differential Effects of Myo-Inositol Pawels Kurian, 1 Neelam Narang, 1 L. Judson Chandler, 1 and Fulton T. Crews 1,2 (Accepted October 5, 1992) To investigate the effects of increasing concentrations of myo-inositol (inositol) on receptor stim- ulated [3H]inositol polyphosphate formation in the absence of lithium, slices of rat cerebral cortex were incubated with various concentrations of [3H]inositol (1 to 30 IxM). Carbachol stimulated formation of [3H]inositol trisphosphate (InsP3) and [3H]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P4} increased several fold when the inositol concentration was increased reaching a plateau at approximately 12 ~M inositol. Time course studies revealed that in the presence of low concentrations of inositol (1 ~M), [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation in response to carbachol stimulation increased slowly over a 10 to 20 rain time period, whereas in the presence of 4 and 12 ~M inositol, carbachol stimulated [3H]InsP3 and [3H]Ins(1,3,4,5)P4 formation was rapid and essentially complete within 3 to 5 rain after carbachol addition. Although the carbachol dose response in 12 IxM inositol had a much greater maximal efficacy, there was no change in potency. Similar to the effects of carbachol on [3H]Ins(1,3,4,5)P4 formation from prelabeled phos- phoinositides, muscarinic receptor stimulation increased Ins(1,3,4,5)P4 mass formation by seven fold. Furthermore, Li § (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P4 mass formation. In contrast to the effects of increasing inositol on carbachol stimulated formation of radiolabeled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P4. These results show that when measuring inositol polyphosphate formation by the radiolabeling technique in the absence of Li§ increasing the inositol concentration greatly increases the stimulated component of [3H]InsP3 and [aH]Ins(1,3,4,5)P4 formation. However, this inositol induced increase in agonist stimulated Ins(1,3,4,5)P4 formation is not reflected as an increase in mass formation. KEY WORDS: Brain; carbachol; inositol; inositol phosphates; inositol tetrakisphosphate; lithium. INTRODUCTION Phosphoinositide hydrolysis is an important signal- ing pathway in a variety of tissues including brain. A Department of Pharmacology, University of Florida College of Med- icine, Gainesville, FL 32610-0267. 2 Address reprint requests to Dr. Fulton T. Crews, Department of Pharmacology, Box 100267, University of Florida College of Med- icine, Gainesville, FL 32610-0267, U.S.A. Ph. (904) 392-2929, FAX (904) 392-0697. 639 number of neurotransmitters and hormones have been shown to stimulate phosphoinositide hydrolysis via ac- tivation of phospholipase C. Hydrolysis of phosphati- dylinositol 4,5-bisphosphate [PtdIns(4,5)P2] results in the formation of two putative second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Ins(1,4,5)P3 mobilizes intracellular calcium from a non-mitochondrial, ATP-dependent pool whereas DAG activates protein kinase C. The metabolism of Ins(1,4,5)P3 may also result in formation of other important second 0364-3190/93/0500-0639507.00/0 9 1993 Plenum Publishing Corporation