,J Mel Crll Cardiol
Guanosine
24, 683-690
(1992)
Metabolism in A&h Rat Cardiac Myocytes: Inhibition Acyclovir and Analysis of a Metabolic Pathway
Timothy
P. Geisbuhler*,
Department
of Physiology,
(Received
1 October
Tamxni
e L. Schwager,
Universit>b 1.991,
qfhfissouri.
accepted
and Michael
Columbia.
in rwi\ed,form
MO
J. Rovetto
65212.
28, January
by
I :X4
1991)
7‘. P. GEISRUHLER,
T. L. SCHWACER AND M. J. Ru\w‘1‘0. Guanosine Metabolism in Adult Rat Cardiac Mvoc ytes: Inhibition by Acyclovir and Analysis of a Metabolic Pathway. LJournnl of Mulecular and Cefluiar Carddop (1992) 24. 683-691, The metabolic fate of transported guanosine was examined in adult rat cardiac myocytt~. Freshly isolated cells were incubated with 50~~ 8-[‘HI-g uanosine and the purine nucleoside phosphwylasc (PNP) inhibitor aryclovir. and the nucleotide products extracted and examinrd for radiolabel distribution Acyclovir inhibited guanosine incorporation into the 5’-nucleotidc pool up to 66%‘. Thr drug did not inhibit guanosine transport. Other experiments using 5’-[ ‘HI-g uanoslne and 8-[‘4C]-guanosine in concert as metabolic tracers showed both tritium and radiocarbon in the guanine nucleotide products. We concluded from this study that both a kinase (probably adenosine kinase) and the enzyme pair purine nucleosidr phosphoryla\eihyp(lxanthine-guanine phosphoryibosyltransferase are rrsponsiblr for guanosine salvage in heart cells. KEY WORDS:
Cardiac
myocytes;
Guanosine;
Ribose;
Introduction Previous work has shown that guanosine, like adenosine, can be metabolized to 5’-nucleotides in the cardiac muscle cell [I]. These metabolic conversions (termed “salvage”) are important to cardiac myocytes, which cannot produce 5’-nucleotides de novo from amino acids and other precursors. The loss of 5’-nucleotides from the heart under ischemic conditions is well documented. Stimulation of myocardial nucleoside salvage under post-ischemic conditions could lead to improved cardiac function. Knowledge of salvage pathways would be essential to developing treatment strategies. Ribose, a precursor of 5-phosphoribosyl-1-pyrophosphate (PRPP), stimulates incorporation of guanosine into 5’-nucleotides. Adenosine metabolism to AMP (which is accomplished via a specific kinase) is unaffected by ribose. This indicates that a metabolic pathway other than adenosine or guanosine kinase is involved in guanosine metabolism to 5’-nucleotides (see Fig. 1). In this study, we examined further how heart *To whom reprint Mcdicinr. ROO W 0022-2828/92/070683
requests ,JctGrson
should Street,
+ 09 $03.00/O
Nucleoside
metabolism
muscle cells incorporate extracellular guanosine into cellular 5’-nucleotides. Vsing acyclovir [an antiviral agent and purinc nucleoside phosphorylase (PNP) inhibitor] along with differentially radiolabellcd guantjsine, we have confirmed the involvement ot PNP in guanosine metabolism. By indircc.r methods, we have also confirmed the activit\ of hypoxanthine-guanine phosphoribcqltransferase (HGPRT) in guanosinc salvage activity. Our results suggest the existence of a dual metabolic pathway for guanosine salvagt into 5’-nucleotides.
Methods isolation
of 77!yo@n
Myocytes were isolated from adult rat hearts as described previously [I]. The ccl1 suspension was examined for rod-shaped cells and viability using 10% glutaraldehydr/O.:%‘X~ trypan blue as described by Hohl et al. [21. The myocytc~ preparations routinely contained 70-90%8 rod shaped cells and >90’$ viable crlls. Stutlies
bc addressed: I)cpartment Kirksvillc. MO6’~iOl. CSA
01 l’hyvolog),
Iiirks\illv
“-‘l!w
(:
Guanosine
24, 683-690
(1992)
Metabolism in A&h Rat Cardiac Myocytes: Inhibition Acyclovir and Analysis of a Metabolic Pathway
Timothy
P. Geisbuhler*,
Department
of Physiology,
(Received
1 October
Tamxni
e L. Schwager,
Universit>b 1.991,
qfhfissouri.
accepted
and Michael
Columbia.
in rwi\ed,form
MO
J. Rovetto
65212.
28, January
by
I :X4
1991)
7‘. P. GEISRUHLER,
T. L. SCHWACER AND M. J. Ru\w‘1‘0. Guanosine Metabolism in Adult Rat Cardiac Mvoc ytes: Inhibition by Acyclovir and Analysis of a Metabolic Pathway. LJournnl of Mulecular and Cefluiar Carddop (1992) 24. 683-691, The metabolic fate of transported guanosine was examined in adult rat cardiac myocytt~. Freshly isolated cells were incubated with 50~~ 8-[‘HI-g uanosine and the purine nucleoside phosphwylasc (PNP) inhibitor aryclovir. and the nucleotide products extracted and examinrd for radiolabel distribution Acyclovir inhibited guanosine incorporation into the 5’-nucleotidc pool up to 66%‘. Thr drug did not inhibit guanosine transport. Other experiments using 5’-[ ‘HI-g uanoslne and 8-[‘4C]-guanosine in concert as metabolic tracers showed both tritium and radiocarbon in the guanine nucleotide products. We concluded from this study that both a kinase (probably adenosine kinase) and the enzyme pair purine nucleosidr phosphoryla\eihyp(lxanthine-guanine phosphoryibosyltransferase are rrsponsiblr for guanosine salvage in heart cells. KEY WORDS:
Cardiac
myocytes;
Guanosine;
Ribose;
Introduction Previous work has shown that guanosine, like adenosine, can be metabolized to 5’-nucleotides in the cardiac muscle cell [I]. These metabolic conversions (termed “salvage”) are important to cardiac myocytes, which cannot produce 5’-nucleotides de novo from amino acids and other precursors. The loss of 5’-nucleotides from the heart under ischemic conditions is well documented. Stimulation of myocardial nucleoside salvage under post-ischemic conditions could lead to improved cardiac function. Knowledge of salvage pathways would be essential to developing treatment strategies. Ribose, a precursor of 5-phosphoribosyl-1-pyrophosphate (PRPP), stimulates incorporation of guanosine into 5’-nucleotides. Adenosine metabolism to AMP (which is accomplished via a specific kinase) is unaffected by ribose. This indicates that a metabolic pathway other than adenosine or guanosine kinase is involved in guanosine metabolism to 5’-nucleotides (see Fig. 1). In this study, we examined further how heart *To whom reprint Mcdicinr. ROO W 0022-2828/92/070683
requests ,JctGrson
should Street,
+ 09 $03.00/O
Nucleoside
metabolism
muscle cells incorporate extracellular guanosine into cellular 5’-nucleotides. Vsing acyclovir [an antiviral agent and purinc nucleoside phosphorylase (PNP) inhibitor] along with differentially radiolabellcd guantjsine, we have confirmed the involvement ot PNP in guanosine metabolism. By indircc.r methods, we have also confirmed the activit\ of hypoxanthine-guanine phosphoribcqltransferase (HGPRT) in guanosinc salvage activity. Our results suggest the existence of a dual metabolic pathway for guanosine salvagt into 5’-nucleotides.
Methods isolation
of 77!yo@n
Myocytes were isolated from adult rat hearts as described previously [I]. The ccl1 suspension was examined for rod-shaped cells and viability using 10% glutaraldehydr/O.:%‘X~ trypan blue as described by Hohl et al. [21. The myocytc~ preparations routinely contained 70-90%8 rod shaped cells and >90’$ viable crlls. Stutlies
bc addressed: I)cpartment Kirksvillc. MO6’~iOl. CSA
01 l’hyvolog),
Iiirks\illv
“-‘l!w
(:
