Aging and Calcitriol Regulation of IP₃ Production in Rat Skeletal Muscle and Intestine

We previously reported that calcitriol [1,25(OH)₂-vitamin D₃] in rat skeletal muscle and duodenum stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP₃) and diacylglycerol (DAG), and that this mechanism is altered in old animals. As previously reported in muscle, we show in the present study that GTPγS (100 μM, 15 s), the non-hydrolyzable analogue of GTP, increased IP₃ release from young rats duodenum to the same extent as 1 nM calcitriol (+ 100 %), while GDPβS (100 μM) suppressed hormone-dependent IP₃ production. Similarly to calcitriol, GTPγS response was diminished in old rats. Contrary to muscle, pretreatment with Bordetella pertussis toxin did not modify calcitriol-dependent IP₃ in duodenum. The antibody, anti-Gαq/11 (1 : 200) and anti-Gαi (1 : 200) blocked calcitriol-dependent IP₃ release in muscle from young rats, indicating that the hormone activates an isoform of PLC coupled to the α subunit of Gq/11 and possibly the βγ subunits of Gi. The aged muscle was insensitive to anti Gαi. In rat duodenum the hormone effects were suppressed by anti-Gq/11 both in young and aged animals. In 24-month-old rats, Gq/11 and Gi protein levels were greatly reduced both in muscle and duodenum, suggesting that a deficiency in G protein expression with aging may have important consequences for correct receptor/effector coupling and could explain age-related declines in the function of second messenger systems linked to G-proteins.

Key words:

Calcitriol - IP₃ - Rat Muscle - Rat Duodenum - Aging - Signal Transduction

References

• 1 Norman A W, Putkey J A, Nemere I. Intestinal calcium transport: pleiotropic effects mediated by vitamin D.  Fed Proc. 1982;  41 78-83
• 2 Reichel H, Koeffler H P, Norman A W. The role of the vitamin D endocrine system in health and disease.  N Engl J Med. 1989;  20 980-991
• 3 Koeffler H P. Vitamin D: myeloid differentiation and proliferation.  Haematol Blood Transfus. 1985;  29 409-417
• 4 Pols H A, Birkenhager J C, Foekens J A, van Leeuwen J P. Vitamin D: a modulator of cell proliferation and differentiation.  J Steroid Biochem Mol Biol. 1990;  37 873-876
• 5 DeLuca H F, Heinrich K, Schnoes A. Vitamin D: recent advances.  Ann Rev Biochem. 1983;  52 411-439
• 6 Minghetti P P, Norman A W. 1,25(OH)₂-vitamin D₃ receptors: gene regulation and genetic circuit.  FASEB J. 1988;  2 3043-3053
• 7 Boland A R, Nemere I. Rapid actions of Vitamin D compounds.  J Cell Biochem. 1992;  49 32-36
• 8 Walters M R. Newly identified actions of the vitamin D endocrine system.  Endocr Rev. 1992;  13 719-764
• 9 Fleet J C. Vitamin D receptors: not just in the nucleus anymore.  Nutr Rev. 1999;  57 60-62
• 10 Boland A R, de Flawia M, Coso O, Boland R. A guanine nucleotide-binding protein mediates 1,25-dihydroxyvitamin D₃-dependent rapid stimulation of Ca²⁺ uptake in skeletal muscle.  Acta Biochim Biophys. 1991;  1094 238-242
• 11 Boland A R, de Morelli S, Boland R. 1,25(OH)₂-Vitamin D₃ stimulates phospholipase A₂ activity via a guanine nucleotide-binding protein in chick myoblasts.  Biochim Biophys Acta. 1995;  1257 274-278
• 12 Morelli S, Boland  R, Boland A R. 1,25(OH)₂-Vitamin D₃ stimulation of phospholipases C and D in muscle cells involves extracellular calcium and a pertussis-sensitive G protein.  Mol Cell Endocrinol. 1996;  122 207-211
• 13 Boland de A R, Facchinetti M M, Balogh G, Massheimer V, Boland R. Age-associated decrease in inositol 1,4,5-trisphosphate and diacylglycerol generation by 1,25(OH)₂-vitamin D₃ in rat intestine.  Cell Signal. 1996;  8 153-157
• 14 Facchinetti M M, Boland R, Boland de A R. Age-related loss of calcitriol stimulation of phosphoinositide hydrolysis in rat skeletal muscle.  Mol Cell Endoclinol. 1998;  136 131-138
• 15 Facchinetti M M, Boland de A R. Effect of ageing on the expression of protein kinase C and its activation by 1,25(OH)₂-vitamin D₃ in rat skeletal muscle.  Cell Signal. 1999;  11 39-44
• 16 Marinissen M J, Selles J, Boland R. Involvement of protein kinase C in 1,25(OH)₂-vitamin D₃ regulation of calcium uptake by cultured myocytes.  Cell Signal. 1994;  6 531-538
• 17 Balogh G; Boland de AR; Boland R. Influence of age on 1,25(OH)₂-vitamin D₃ activation of protein kinase C in rat duodenum.  Molec and Cell Endocrinol. 1997;  129 127-133
• 18 Vazquez G, de Boland A R. Involvement of protein kinase C in the modulation of 1alpha,25-dihydroxy-vitamin D₃-induced ⁴⁵Ca²⁺ uptake in rat and chick cultured myoblasts.  Biochim Biophys Acta. 1996;  1310 157-162
• 19 Vazquez G, de Boland A R, Boland R L. 1alpha,25-(OH)₂-Vitamin D₃ stimulates the adenylyl cyclase pathway in muscle cells by a GTP-dependent mechanism which presumably involves phosphorylation of G alpha i.  Biochem Biophys Res Commun. 1997;  234 125-128
• 20 Vazquez G, de Boland A R, Boland R L. Stimulation of Ca²⁺ release-activated Ca²⁺ channels as a potential mechanism involved in non-genomic 1,25(OH)₂-vitamin D₃-induced Ca²⁺ entry in skeletal muscle cells.  Biochem Biophys Res Commun. 1997;  239 239, 562-565
• 21 Vazquez G, de Boland A R, Vazquez G, Boland R. 1alpha,25-Dihydroxy-vitamin-D₃-induced store-operated Ca²⁺ influx in skeletal muscle cells. Modulation by phospholipase C, protein kinase C, and tyrosine kinase.  J Biol Chem. 1998;  273 33 954- 33 960
• 22 Norman A W, Song X D, Ishizuka S, Bula C, Bishop J, Okamura W H. 1α,25(OH)₂D₃-mediated rapid and genomic responses in human leukemic NB4 cells: Evidence for involvement of crosstalk from rapid responses to genomic effects. In: Norman AW, Buillon R, Thomasset M (eds). Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects. University of California Press ( in press)
• 23 Massheimer V, Boland R, de Boland A R. Rapid 1,25(OH)₂-vitamin D₃-stimulation of calcium uptake by rat intestinal cells involves a dihydropyridine-sensitive cAMP-dependent pathway.  Cell Signal. 1994;  6 299-304
• 24 Massheimer V, Picotto G, de Boland A R, Boland R. Aging alters the rapid stimulation of cAMP-dependent calcium uptake by 1,25(OH)₂-vitamin D₃ in rat intestinal cells.  Endocr and Metabolism. 1995;  2 157-163
• 25 Balogh G A, de Boland A R. 1,25-Dihydroxy-vitamin D₃ (calcitriol)-dependent protein phosphorylation in rat duodenum: effects of aging.  Experimental Gerontology. 1999;  34 983-996
• 26 Lowry O H, Rosehrrough, NJ, Farr A L, Randall R J. Protein measurement with the Folin phenol reagent.  J Biol Chem. 1951;  193 265-275
• 27 Bredt D S, Mourey R J, Snyder S H. A simple, sensitive, and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues.  Biochem Biophys Res Commun. 1989;  159 976-982
• 28 Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-685
• 29 Snedecor G W, Cochran W G. Statical Methods.  Ames, IA; The Iowa State University Press 1967
• 30 Stokes C E, Gillon K RW, Hawthorne J N. Free and total lipid myo-inositol concentrations decrease with age in human brain.  Biochim Biophys Acta. 1983;  753 136-138
• 31 Martini A, Battaini F, Govoni S, Volpe P. Inositol 1,4,5-trisphosphate receptor and ryanodine receptor in the aging brain of Wistar rats.  Neurobiol Aging. 1994;  15 203-206
• 32 Mizutani T, Nakashima S, Nozawa Y. Changes in the expression of protein kinase C (PKC) and phospholipases C (PLC) and D (PLD) isoforms in spleen, brain and kidney of the aged rat: RT-PCR and Westen Blot analysis.  Mech Ageing Dev. 1998;  105 151-172
• 33 Fulop T, Foris G, Worum Y, Leovey A. Age-dependent alterations of Fc gamma receptor-mediated effector functions of human polymorphonuclear leucocytes.  Clin Exp lmmunol. 1985;  61 425-432
• 34 Fulop T, Barabas G, Varga Z, Jozsef C, Csabina S, Szucs S, Seres I, Szikszay E, Jeney Z, Peniyge A. Age-dependent changes in transmembrane signaling: Identification of G proteins in human lymphocytes and polymorphonuclear leukocytes.  Cell Signal. 1993;  5 593-603
• 35 Bastyr E J, Kadrofske M M, Vinik A l. Platelet activity and phosphoinositide turnover increase with advancing age.  Am J Med. 1990;  88 601-606
• 36 Sugawa M. Alterations of inositol phosphate turnover in stratium of aged rats.  Eur J Pharmacol. 1991;  247 39-44
• 37 Roth G S. Changes in tissue responsiveness to hormones and neurotransmitters during aging.  Exp Gerontol. 1995;  30 361-368
• 38 Narang N, Joseph J A, Ayyagari P V, Gerber M, Crews F T. Age-related loss of cholinergic-muscarinic coupling to PLC: comparison with changes in brain regional PLC subtypes mRNA distribution.  Brain Res. 1996;  708 143-152
• 39 Massheimer V, Picotto G, Boland R, de Boland A R. Effect of aging on the mechanisms of PTH-induced calcium influx in rat intestinal cells.  J Cell Physiol. 2000;  182 429-437
• 40 Gilman A G. G-proteins: transducers of receptor-generated signals.  Annu Rev Biochem. 1987;  56 615-649
• 41 Exton J H. Cell signaling through guanine-nucleotide-binding proteins (G-proteins) and phospholipase.  Eur J Biochem. 1997;  243 10-20
• 42 Lierberherr M, Grosse B. Androgens increase intracellular calcium concentration and inositol 1,4,5-trisphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein.  J Biol Chem. 1994;  269 7217-7223
• 43 Katan M. Families of phosphoinositide-specific phospholipase C: Structure and function.  Biochim Biophys Acta. 1998;  46 5-17
• 44 Bairoch A, Cox J A. EF-hand motifs in inositol phospholipid-specific phospholipase C.  FEBS Lett. 1990;  269 454-456
• 45 Ueda H, Morishita R, Katoh-Sembra R, Kato K, Asano T. G-protein γ subunits coimmunoprecipitated with antibodies against subunits: Identifications of major isoforms in cultured cells by silver stain and immunoblotting with conventional transfer procedure.  J Biochem. 1998;  124 1033-1037
• 46 Heron D S, Shinitzky M, Hershkowitz M, Samuel D. Lipid fluidity markedly modulates the binding of serotonin to mouse brain membranes.  Proc Natl Acad Sci USA. 1980;  77 7463-7467
• 47 Yeagle P L, Rice D, Young J. Effects of cholesterol on (Na⁺,K⁺)-ATPase ATP hydrolysing activity in bovine kidney.  Biochemistry. 1988;  27 6449-6452
• 48 Guderman T, Kalkbrenner F, Schultz G. Diversity and selectivity of receptor-G protein interaction.  Annu Rev Pharmacol Toxicol. 1996;  36 429-459
• 49 Johnson M D, Zhou Y, Friedman E, Roberts J. Expression of G protein alpha subunits in the aging cardiovascular system.  J Gerontol A Biol Sci Med Sci. 1995;  50 B14-B19
• 50 Mader S L, Downing C L, Amos-Landgraf J, Swebjka P. Age-related changes in G proteins in rat aorta.  J Gerontol A Biol Sci Med Sci. 1996;  51 B111-B116
• 51 Miyamoto A, Kawana S, Kimura H, Ohshika H. Impaired expression of Gs alpha protein mRNA in rat ventricular myocardium with aging.  Eur J Pharmacol. 1994;  266 147-154
• 52 Hanai H, Liang C T, Cheng L, Sacktor B. Desensitization to parathyroid horone in renal cells from aged rats is associated with alterations in G-protein activity.  J Clin Inv. 1989;  83 268-277
• 53 Parekh V, Maier K G, Roman R J, Joshua I G, Falcone J C, Passmore J C. Altered expression and activity of G-proteins, mitogen activated protein kinases, and tyrosine kinases in aging kidney cortex.  J lnvestig Med. 1999;  47 462-467
• 54 Sawaki K, Baum B J, Roth G S, Ambudkar I S. Decreased m3-muscarinic and alpha 1-adrenergic receptor stimulation of PIP2 hydrolysis in parotid gland membranes from aged rats: defect in activation of Galpha q/11.  Arch Biochem Biophys. 1995;  322 319-326
• 55 Takamoto S, Seino Y, Saktor B, Liang C T. Effect of age on duodenal 1,25(OH)₂-vitamin D₃ receptor in Wistar rats.  Biochim Biophys Acta. 1990;  1034 22-28