Peptides 26 (2005) 691–700

Distribution of atrial natriuretic peptide and its effects on contractionand intracellular calcium in ventricular myocytes

from streptozotocin-induced diabetic rat

F.C. Howartha,∗, A. Adema, E.A. Adeghatea, N.A. Al Ali a,A.M. Al Bastakia, F.R. Soroura, R.O. Hammoudia,

N.A. Ghaleba, N.J. Chandlerb, H. Dobrzynskib

a Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, U.A.E.b School of Biomedical Sciences, University of Leeds, Leeds, UK

Received 19 September 2004; received in revised form 30 November 2004; accepted 2 December 2004Available online 11 January 2005

Abstract

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The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte cond intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood poncentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to agontrols. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myercentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-

he percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myoTZ-treated rats (360± 5 ms) compared to controls (305± 5 ms). Amplitude of the Ca2+ transient was significantly increased in myocy

rom STZ-treated rats compared to controls (0.39± 0.02 versus 0.29± 0.02 fura-2 RU in controls) and treatment with ANP reducedmplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.2004 Elsevier Inc. All rights reserved.

eywords:Atrial natriuretic peptide; Diabetes; Ventricular myocytes; Calcium

. Introduction

Atrial natriuretic peptide (ANP) is a hormone which iseleased predominantly by atrial myocytes in response totrial wall stretch[8]. In the normal healthy adult heart ANP

s mainly synthesized in atrial granules as a larger moleculareight precursor which is believed to be rapidly converted

o an active peptide during or shortly after secretion[32].evels of ANP are increased during experimental hypoxiand in various disease states including hypertension,oronary heart failure, myocardial infarction and diabetes

∗ Corresponding author. Tel.: +971 3 7039536; fax: +971 3 7671966.E-mail address:chris.howarth@uaeu.ac.ae (F.C. Howarth).

[16,28]. ANP induces relaxation of vascular smooth musdecreases blood pressure and cardiac output andactions are associated with inhibition of aldosterone, rand vasopressin release[1]. Ventricular hypertrophy icharacterized by an augmentation of the synthesisrelease of ANP from the ventricles[17]. Treatment oyoung adult rats with streptozotocin (STZ) causes damto pancreatic�-cells, hypoinsulinemia and an associahyperglycemia features which are frequently observeclinical type 1 diabetes[9]. The remodeling of distribution oANP in both atrial and ventricular myocytes and the effof ANP on ventricular myocyte contraction and [Ca2+]i inthe STZ-induced diabetic rat have been investigated instudy.

196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2004.12.003

692 F.C. Howarth et al. / Peptides 26 (2005) 691–700

2. Materials and methods

2.1. Induction of diabetes

Diabetes was induced by a single intraperitoneal injectionof STZ (60 mg/kg; Sigma) administered to young male Wistarrats (200–250 g; bred in-house). The STZ was dissolved in acitrate buffer solution (0.1 mol/l citric acid, 0.1 mol/l sodiumcitrate; pH 4.5). Age-matched controls received an equiva-lent volume of the citrate buffer solution alone. Both groupsof animals were maintained on the same diet and water adlibitum until they were used 8–12 weeks later. Principles oflaboratory animal care were followed throughout. Approvalfor this project was obtained from the Faculty of Medicineand Health Sciences Ethics Committee.

2.2. Measurement of ANP in blood plasma

ANP radioimmunoassay was performed using previouslydescribed techniques[22,25]. Briefly, blood samples werecollected in disodium ethylenediaminetetraacetic acidvacutainers with added aprotinin (500 KIU/ml of blood) andcentrifuged at 1600×g for 15 min at 4◦C. Plasma sampleswere stored at−80◦C until assayed for ANP. Cardiac tissuewas weighed and homogenized in 2 ml of phosphate buffereds 0a say.C aftere with1 ilei ess,t ANPi oas-s elsi werem

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singp th e tis-s ti erel ared

in xylene and embedded in paraffin wax. Longitudinal sec-tions of 6�m thickness across atrial and ventricular muscleswere cut and deparaffinized in xylene. The sections werethen incubated for 30 min in 0.3% hydrogen peroxide so-lution in methanol to block endogenous peroxidase activ-ity. The sections were washed three times (5 min each) inTris-buffered saline (TBS) and incubated in the blockingreagent for 30 min. The samples were later incubated in poly-clonal antibodies against ANP (1:1000 dilution) 24 h at 4◦Cfollowed by 30 min incubation in pre-diluted biotinylatedanti-rabbit IgG. After washing in TBS the specimens wereincubated in streptavidin peroxidase conjugate for 45 minand treated with 3,3-diaminobenzidine tetra-hydrochlorideto reveal sites of immunoreactivity. The slides were counter-stained with hematoxylin for 30 s, dehydrated in ascendinggrades of ethanol, cleared in xylene and mounted in Cytoseal60 (Stephens Scientific, USA).

2.5. Distribution of ANP in ventricular myocytes

Immunocytochemistry experiments were carried out ac-cording to previously described techniques[20]. Freshly iso-lated ventricular myocytes were plated on polylysine coatedslides (BDH; Poole, UK) and allowed to settle for 45 min.Cells were then fixed in 2% paraformaldehyde and washedt in.C PBSc reet ith1 be-f DSa eb PBSo don-k keys mi-c withP ato-r clearn tv cro-s hent nots my-o nningc r ofA untedu

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ingtk nga on aL ely at

aline (PBS). Homogenates were centrifuged at 20,00×gnd 100�l of the supernatant was used for ANP asoncentrations of ANP were determined in plasmaxtraction on a Sep-Pak C18 column, pre-equilibrated% trifluoroacetic acid. After elution with 60% acetonitr

n 1% trifluoroacetic acid and evaporation to drynhe residue was reconstituted in assay buffer andmmunoreactivity measured with a specific radioimmunay kit (Peninsula Laboratories, CA, USA). ANP levn plasma were expressed as pg/ml. Protein levels

easured using the Bradford dye-binding procedure[4].

.3. Measurement of ANP in atria and ventricles

Heart atria and ventricles were weighed and homogenn 2 ml of PBS. Homogenates were centrifuged at 20,000×gnd 100�l of the supernatant was used for ANP assay. Aadioimmunoassay was performed as described above upecific radioimmunoassay kit (Peninsula Laboratories,SA). ANP levels in heart atria and ventricles were expres pg/mg protein. Protein levels were measured usinradford dye-binding procedure[4].

.4. Distribution of ANP in cardiac muscle

Rat heart immunohistochemistry was performed ureviously described techniques[22]. Briefly, whole raearts were trimmed free of adherent fat and connectivue and cut into small pieces (2 mm3) and fixed overnigh

n freshly prepared Zamboni’s fixative. The specimens water dehydrated in graded concentrations of ethanol, cle

hree times with 0.01 M PBS (Sigma, Poole, UK) over 30 mells were then permeabilized by incubating them inontaining 0.2% Triton X-100 for 30 min and washed thimes with PBS. Blocking was carried out for 30 min w0% normal donkey serum (NDS) and 1% BSA in PBS

ore incubation with primary antibodies (diluted in 10% Nnd 1% BSA in PBS) overnight at 4◦C. The slides werrought to room temperature, washed three times withver 30 min, and incubated with secondary antibody,ey anti-mouse IgG (1:100; diluted in 10% normal donerum and 1% BSA in PBS) conjugated to FITC (Cheon) for 60 min. Cells were washed a further three timesBS, mounted with Vectashield (H-1000; Vector Labor

ies, Peterborough, UK) and the coverslips sealed withail polish, and stored in the dark at 4◦C for subsequeniewing in a Leica TCS SP laser scanning confocal micope. No labeling above background was obtained whe primary or secondary antibodies were omitted (datahown). Images of ANP expressing and non-expressingcytes were collected using a Leica TCS SP laser scaonfocal microscope (Leica Microsystems). The numbeNP expressing and non-expressing myocytes was conder a Leica epifluorescent microscope.

.6. Ventricular myocytes isolation

Single ventricular myocytes were isolated accordo previously described technique[13]. In brief, rats wereilled humanely by cervical dislocation following stunnind their hearts were removed quickly and mountedangendorff apparatus. Hearts were perfused retrograd

F.C. Howarth et al. / Peptides 26 (2005) 691–700 693

a constant flow of 8 ml/g heart−1 min−1 with a HEPES-basedisolation solution (see below) containing 0.75 mM Ca2+. Per-fusion flow rate was adjusted to allow for differences in heartweight between STZ-treated and control animals. When thecoronary circulation had cleared of blood, perfusion was con-tinued for 4 min with a Ca2+-free isolation solution containing0.1 mM EGTA, and then for 6 min with solution containing0.05 mM Ca2+, 0.75 mg/ml collagenase (type 2; Worthing-ton, N.J., USA) and 0.075 mg/ml protease (type X1V;Sigma). After this time, the ventricles were excised from theheart, minced and gently shaken in collagenase-containingisolation solution supplemented with 1% BSA. Cells werefiltered from this solution at 4 min intervals and resuspendedin 0.75 mM Ca2+-containing isolation solution. Cell viability,defined as the percentage of rod-shaped myocytes in the cellsuspension was recorded within 1 h of completing the cellisolation.

2.7. Ventricular myocyte experiments

Freshly isolated myocytes suspended in 0.75 mM Ca2+-containing isolation solution were divided into two aliquots,centrifuged at low speed and the cell pellets resuspendedeither in normal Tyrode (see below) or normal Tyrodecontaining 10−7 M or 10−6 M ANP (Sigma, A-8208). Cellsw t ofe ytesw spexc cope( sed( tioncA tedmm etec-t e ofs ngth)t k toh aw atorf bedp -t nma arch,E Ther by a

photomultiplier tube and the ratio of the emitted fluorescenceat the two excitation wavelengths (340/380 ratio) was calcu-lated to provide an index of intracellular Ca2+ concentration.

2.8. Solutions

The cell isolation solution contained (in mM) 130.0 NaCl,5.4 KCl, 1.4 MgCl2, 0.4 NaH2PO4, 5 HEPES, 10 glucose, 20taurine and 10 creatine set to pH 7.3 with NaOH. The normalTyrode solution contained (in mM): NaCl 140; KCl 5; MgCl21; glucose 10; HEPES 5; CaCl2 1 set to pH 7.4 with NaOH.

2.9. Statistics

Results were expressed as the mean± S.E.M. of ‘n’ ob-servations. ‘n’ refers either to the number of rats or number ofcells. Statistical comparisons were performed using either in-dependent samplest-test or ANOVA followed by Bonferronicorrectedt-tests for multiple comparisons, as appropriate.P< 0.05 was considered to indicate a significant difference.Statistical analysis was carried out using either SigmaStat(Jandel Scientific, USA) or SPSS (USA).

3. Results

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ere incubated in ANP for 1 h before commencemenxperiments. For electrophysiological studies myocere allowed to settle on the glass bottom of a Perhamber mounted on the stage of an inverted microsAxiovert 35, Zeiss, Germany). Myocytes were superfu3–5 ml/min) with a HEPES-based normal Tyrode soluontaining 1 mM Ca2+ or normal Tyrode containing 10−7 MNP. Experiments were performed in electrically stimulayocytes (1 Hz) at room temperature (23–25◦C). Unloadedyocytes shortening was followed using a video edge d

ion system (VED-114, Crystal Biotech, USA). The degrehortening (expressed as a percentage of resting cell lehe time to peak shortening (TPK) and time from peaalf relaxation (THALF) were recorded. Intracellular C2+

as measured in cells loaded with the fluorescent indicura-2 AM (F-1221, Molecular Probes, USA) as descrireviously [11]. To measure intracellular Ca2+ concen

ration, myocytes were alternately illuminated by 340nd 380 nm light using a monochromator (Cairn Resengland) which changed the excitation light every 2 ms.

esultant fluorescent emission at 510 nm was recorded

able 1eneral characteristics of control and STZ-treated rats

ody weight (g)eart weight (g)eart weight/body weight ratio (mg/g)lood glucose (mg/dl)

ata are mean± S.E.M.∗∗ P< 0.01, independent samplest-test. Numbers of animals are shown

,

.1. General characteristics of STZ-induced diabetic ra

The general characteristics of STZ-treated rats compith their age-matched controls are shown inTable 1. Di-betes was confirmed in STZ- treated rats by a signifi-fold, elevation of blood glucose. STZ-treated rats cha

eristically had significantly lower body weights and heeights compared with controls. Heart weight/body we

atio was slightly increased in STZ-treated rats comparge-matched controls.

.2. Levels of ANP in blood plasma and its distributionn cardiac muscle

The concentrations of ANP in blood plasma as wen atrial and ventricular muscles are shown inFig. 1. Theoncentration of ANP was significantly (P< 0.05) increasen blood plasma (a) and in atrial (b) and ventricular

uscles after STZ treatment. The concentration of ANlood plasma from STZ-treated rats was 124.75± 8.37 pg/m

ntrol STZ

0.6± 16.2 (5) 224.0± 25.6 (5)**

.25± 0.05 (4) 1.02± 0.05 (5)**

4.28± 0.41 (4) 4.71± 0.40 (5)68.0± 2.3 (5) 347.4± 15.2 (5)**

ntheses.

694 F.C. Howarth et al. / Peptides 26 (2005) 691–700

Fig. 1. Quantification of ANP in blood plasma and cardiac muscle. Meanconcentrations of ANP in blood plasma (a), and content of ANP in atrialmuscle (b) and ventricular muscle (c) from STZ-treated and age-matchedcontrol rats. Data are mean± S.E.M.,n= 3 rats for each group. Means sta-tistically compared using Independent samplest-test,*P< 0.05.

(n= 3) compared to 78.67± 4.07 pg/ml (n= 3) in control rats.The level of ANP in atrial muscle from STZ-treated ratswas 2677.0± 287.5 pg/mg protein (n= 3 hearts) comparedto 1524.7± 127.9 pg/mg protein (n= 3 hearts) in control rats.The level of ANP in ventricular muscle from STZ-treated ratwas 291.7± 43.6 pg/mg protein (n= 3 hearts) compared to132.7± 6.4 pg/mg protein (n= 3 hearts) in controls.Fig. 2shows typical micrographs of ANP distribution in the atrialmuscle from control (a) and STZ-treated (b) rats. A clear in-crease in ANP labeling can be seen in cardiac muscle fromSTZ-treated rat.

Representative immunofluorescence confocal micro-graphs showing the expression of ANP and a graph showingthe distribution of ANP in ventricular myocytes from con-trol and STZ-treated rats are shown inFig. 3. Myocytes fromcontrol rats that showed positive ANP labeling and nega-tive ANP labeling are shown in (a) and (b). Myocytes fromSTZ-treated rats that have positive ANP labeling and nega-tive ANP labeling are shown in (c) and (d). The percentage ofcells that have positive or negative ANP labeling are shownin (e). The percentage of ANP(+) and ANP(−) cells in my-ocytes from control rat were 16.8± 5.3 (n= 67 cells) and83.10± 5.3% (n= 67 cells), respectively. The percentage ofANP(+) and ANP(−) cells in myocytes from STZ-treated ratwere 52.1± 7.2 and 53.2± 4.6% (n= 67), respectively.

3m

g aL a Mi-c ocyte

Fig. 2. Distribution of ANP in immunolabeled cardiac muscle. Typical micr l (a) andSTZ-treated (b) rats. The micrographs are typical of 3 such hearts from cont

.3. Characteristics of ventricular myocytes andyocytes shortening

Images of ventricular myocytes were collected usineica TCS SP laser scanning confocal microscope (Leicrosystems), and the surface area of each ventricular my

ographs to show the pattern of distribution of ANP in atria of controrol and diabetic rats. Scale bar = 50�m.

F.C. Howarth et al. / Peptides 26 (2005) 691–700 695

Fig. 3. Distribution of ANP in immunolabeled ventricular myocytes. Representative projection immunoconfocal micrographs to show the expression of ANPlabeling in rat ventricular myocytes from control (a, b) and from STZ-treated rats (c, d). Mean data showing the percentage of ANP positively immunolabeledand ANP negatively immunolabeled ventricular myocytes in STZ-treated compared to control rats (e). Data are mean± S.E.M.,n= 67 from 3 control and 3diabetic rat hearts. Means statistically compared using ANOVA followed by Bonferroni correctedt-tests,+P< 0.05.

696 F.C. Howarth et al. / Peptides 26 (2005) 691–700

Fig. 4. Effects of STZ-induced diabetes on the surface area of ventricularmyocytes. Mean data to show surface area of ventricular myocytes. Dataare mean± S.E.M.,n= 260–273 cellsn= 67 from 3 control and 3 diabeticrat hearts. Means statistically compared using independent samplest-test,*P< 0.05.

was measured using Scion Image (NIH, Bethesda, MD). Thesurface area of myocytes from STZ-treated rat was signifi-cantly (P< 0.05) greater than that of controls confirming thatSTZ-induced diabetes is characterized by hypertrophy of theventricular myocardium (Fig. 4).

A typical fast-time base recording of shortening in anelectrically stimulated (1 Hz) myocyte superfused with nor-mal Tyrode containing 1 mM Ca2+ from a control rat isshown inFig. 5a. Electrophysiological data was acquired inmyocytes from 3–5 control or STZ-treated rats. The meanRCL of myocytes from STZ-treated (123± 5�m, n= 19)was marginally greater than that of controls (117± 4�m,n= 27) however, the difference did not reach statistical sig-nificance (P> 0.05). Based on a previous study which in-vestigated the effects of ANP at different concentrations onfractional myocyte shortening we selected a concentration of10−7 M ANP for this study[33]. Exposure to 10−7 M ANPfor at least 1 h did not significantly alter RCL. The time courseof contraction was significantly (P< 0.05) prolonged in my-ocytes from STZ-treated rat compared to controls (Fig. 5b).The mean TPK shortening was 360± 5 (n= 19) in my-ocytes from STZ-treated rats compared to 305± 5 ms (n= 27)from age-matched controls (Fig. 5b). Exposure of cells with10−7 M ANP had no significant additional effect on TPKshortening in myocytes from either STZ-treated or controlr redbT ge ofR ted(n i-tc -p fromS dt

3.4. Characteristics of ventricular myocyte Ca2+

transient

Typical fast-time base recordings of Ca2+ transients in my-ocytes from an STZ-treated rat superfused with either nor-mal Tyrode or normal Tyrode containing 10−7 M ANP areshown inFig. 6a. Electrophysiological data was acquired inmyocytes from 3–5 control or STZ-treated rats. The meanresting fura-2 ratio in myocytes from STZ-treated rats wassignificantly (P< 0.05) higher (2.31± 0.04 RU,n= 20 cells)compared to that in controls (2.10± 0.06 RU,n= 26 cells)suggesting that resting Ca2+ concentration was elevated inmyocytes from STZ-treated rat. Exposure to 10−7 M ANPfor at least 1 h did not significantly (P> 0.05) alter the restingfura-2 ratio.

The time course of the Ca2+ transient was signifi-cantly (P< 0.05) prolonged in myocytes from STZ-treatedrat compared to controls (Fig. 6b and c). In particularthe mean THALF relaxation of the Ca2+ transient was267± 7 (n= 20) in myocytes from STZ-treated rats com-pared to 215± 9 ms (n= 26) in controls (Fig. 6c). Expo-sure of cells to 10−7 M ANP had no additional significant(P> 0.05) effect on THALF relaxation. The TPK ampli-tude of the Ca2+ transient was slightly increased in my-ocytes from STZ-treated rats and was additionally prolongedb thedT -cnId sf tw ols(

4

sclea on-t icr s notp hoicef sa d re-d easei stentw ra-t con-sT witha areaa cytesi phy[

ats. The THALF relaxation of contraction was not altey either STZ-treatment or by exposure to ANP (Fig. 5c).he amplitude of shortening, expressed as a percentaCL, was slightly increased in myocytes from STZ-trea

9.6± 0.5%,n= 19) rat compared to controls (9.3± 0.5%,= 27). Exposure to 10−7 M ANP slightly reduced ampl

ude of shortening in myocytes from STZ-treated (∼8%) andontrol (∼3%) rats (Fig. 5d), and further reductions in amlitude of shortening were observed when myocytesTZ-treated (∼15%) and control (∼15%) rats were expose

o 10−6 M ANP.

y exposure to ANP compared to controls however,ifference did not reach statistical significance (Fig. 6b).he amplitude of the Ca2+ transient was significantly inreased in myocytes from STZ-treated (0.39± 0.02 RU,= 20) rat compared to controls (0.29± 0.02 RU,n= 26).

nterestingly, exposure to ANP significantly (P< 0.05) re-uced the amplitude of the Ca2+ transient in myocyte

rom STZ-treated rats (0.28± 0.04 RU,n= 17) so that ias no longer significantly different from that of contr

Fig. 6d).

. Discussion

The levels of ANP in blood plasma and cardiac mus well as the effects of ANP on ventricular myocytes c

raction and intracellular Ca2+ in the STZ-induced diabetat have been investigated. This model of diabetes doeroduce atherosclerosis and therefore, is an excellent c

or the study of diabetic cardiomyopathy[2]. At 8–12 weekfter the induction of diabetes we observed a markeuction in body and heart weight and a marked incr

n blood glucose level, characteristics which are consiith the diabetic state. The Heart weight/body weight

io was increased in STZ-treated rat, a finding that isistent with data from several previous studies[12,13,21].his increase in heart weight/body weight ratio togethersignificant increase in ventricular myocyte surface

s well as increased number of ANP expressing myos consistent with STZ-induced cardiac muscle hypertro31].

F.C. Howarth et al. / Peptides 26 (2005) 691–700 697

Fig. 5. Effects of ANP on ventricular myocytes contraction. Representative fast time-base recording of unloaded shortening in a ventricular myocyte isolatedfrom a control rat (a). Mean values of TPK shortening (b), THALF relaxation of contraction (c) and amplitude of shortening (d) in myocytes from STZ-treatedor control rats superfused with either normal Tyrode or normal Tyrode containing 10−7 or 10−6 M ANP. Data are mean± S.E.M.,n= 19–27 from 3–5 controland diabetic rat hearts. Means statistically compared using ANOVA followed by Bonferroni correctedt-tests,+P< 0.05.

Diabetes mellitus results in a fluid and electrolyte imbal-ance which in turn affects blood volume and blood pressure[10]. Our results have shown that ANP concentration in bloodplasma was significantly increased in STZ-treated rats. Pre-vious experiments after different periods of STZ-treatmentranging from 1 week to 8 months have also demonstratedincreases in plasma ANP concentrations[6,22,27,29,30,34].The ANP content of atrial muscle in this study was signif-icantly increased in STZ-treated rats compared to controls.Interestingly, after much shorter periods of STZ treatment (2or 4 weeks), ANP has been shown to decrease in atrial ANP[34]. The ANP content of ventricular muscle in our studywas also significantly increased in STZ-treated rats however,the ANP content of ventricular muscle was a fraction of thatobserved in the atria. This finding is consistent with previ-ous data showing that ANP is produced and stored mainlyby atrial myocytes[8,32]. Increased blood volume increasesboth atrial and ventricular ANP secretions[23,24]. In addi-tion, increased blood pressure (afterload) can also increaseANP secretion from the heart[14,15]. Stretch is believedto be a common haemodynamic stimulus for the synthesis

and release of ANP within the heart[26]. Wu et al. (1998)showed that at 2 or 4 weeks after STZ-treatment concentra-tions of ANP in the plasma and ventricles increased and thatplasma ANP concentrations and ventricular ANP contentswere reversed to normal level by insulin treatment. Wu et al.(1998) also demonstrated a significant increase in the pre-proANP mRNA expression in ventricles but not in the atria.Collectively, these data suggest that STZ-induced diabetesincreases the synthesis and subsequent release of ANP byatrial and ventricular muscles. Thus the increase in plasmaANP concentration is at least in part due to the increased syn-thesis and release of ANP from the atria and ventricles of theheart.

Immunolabeling/confocal techniques were used to furtherelucidate the effects of STZ-induced diabetes on the distri-bution of ANP in atrial and ventricular myocytes. The per-centage of ANP negatively immunolabeled myocytes (i.e.,cells that did not contain ANP immunoreactive binding sites)was relatively high in myocytes from control rats. However,the number of ANP negatively immunolabeled myocyteswas markedly reduced and the number of ANP positively

698 F.C. Howarth et al. / Peptides 26 (2005) 691–700

Fig. 6. Effects of ANP on ventricular myocytes Ca2+ transient. Representative fast time-base recording of Ca2+ transients in ventricular myocytes isolatedfrom an STZ-treated rat superfused with either normal Tyrode or normal Tyrode containing 10−7 M ANP (a). Mean values of TPK Ca2+ transient (b), THALFrelaxation of Ca2+ transient (c) and amplitude of the Ca2+ transient (d) in myocytes from STZ-treated or control rats superfused with either normal Tyrodeor normal Tyrode containing 10−7 M ANP. Data are mean± S.E.M.,n= 17–26 from 3–5 control and diabetic rat hearts. Means statistically compared usingANOVA followed by Bonferroni correctedt-tests,+P< 0.05.

immunolabeled myocytes was markedly increased in my-ocytes from STZ-treated rats. It is interesting to note thatthere is a more than three-fold increase in the number ofANP-expressing cells in ventricular myocardium from STZ-treated rats compared to controls. The increase in the atrialand ventricular contents of ANP may be a protective mech-anism against cardiac hypertrophy which is frequently ob-served in diabetes. These data indicate that in STZ-induceddiabetes there is an upregulation in the distribution of ANP,a finding that is consistent with the reported increase in ANPconcentrations found in cardiac muscle as a whole. ANP hasan important protective role in the heart. For example in pa-tients with congestive heart failure infusion of ANP decreasesafterload and preload and improves left ventricular systolicand diastolic function[19] and in STZ-induced diabetic ratsANP has been shown to reverse cardiac fibrosis[18].

Further experiments were carried out to investigate theeffects of ANP on ventricular myocytes shortening in STZ-treated rats. The time course, and in particular the TPK con-traction, was characteristically prolonged in electrically stim-ulated myocytes from STZ-treated rats compared to controls.

Defects in mechanism(s) of Ca2+ transport including L-typeCa2+ current and impaired release of Ca2+ from the sarcoplas-mic reticulum (SR) may partly underlie these contractiledefects[3,5,7]. TPK shortening was not additionally alteredby ANP. The amplitude of shortening was marginally, thoughnot significantly, increased in myocytes from STZ-treated rat.Exposure of myocytes to ANP caused very small reductionsin amplitude of shortening in myocytes from STZ-treated rats(∼8%) and from controls (∼3%). At higher concentrationsof ANP (10−6 M) there were further small concentration-dependent reductions in the amplitude of shortening in my-ocytes from STZ-treated and control rats. Tajima et al. (1998)showed that the concentration-dependent depression of con-tractility caused by ANP in myocytes from healthy rats maybe attributed to the action of ANP on the Na+/H+ exchangerwhich in turn leads to intracellular acidification and anassociated decrease in myofilament sensitivity to Ca2+ [33].

Experiments were carried out to investigate the effects ofANP on [Ca2+]i in myocytes from STZ-treated rats. The timecourse and in particular the THALF relaxation of the Ca2+

transient was significantly prolonged in electrically stimu-

F.C. Howarth et al. / Peptides 26 (2005) 691–700 699

lated myocytes from STZ-treated rats compared to controls.Defects in mechanism(s) of Ca2+ transport including de-pressed Ca2+ efflux on the Na+/Ca2+ exchanger and impairedSR Ca2+ uptake may partly underlie these contractile defects[7]. THALF relaxation of the Ca2+ transient was not addi-tionally altered by ANP. The TPK Ca2+ transient was slightlyprolonged and additionally prolonged by ANP in myocytesfrom STZ-treated rat compared to controls though the prolon-gations did not reach significance. The amplitude of the Ca2+

transient was significantly increased in myocytes from STZ-treated (0.39± 0.02 RU) compared to control (0.29± 0.02RU) rats. Exposure to ANP caused a marked reduction inamplitude of Ca2+ transient in myocytes from STZ-treatedrats towards the amplitude seen in controls but had little ef-fect in control cells. The mechanisms by which ANP causesa reduction in amplitude of the Ca2+ transient in myocytesfrom STZ-treated rat will require further clarification but mayinclude a reduction in L-type Ca2+ current and/or SR Ca2+

release.

5. Conclusion

The elevations of ANP in blood plasma and heart atrialand ventricular muscles and redistribution of ANP in atriala ts ofA alp t aref per-t

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ex-ytes.

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[8] Christensen G. Release of atrial natriuretic factor. Scand J Clin LabInvest 1993;53:91–100.

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[11] Howarth FC, Calaghan SC, Boyett MR, White E. Effect of themicrotubule polymerizing agent taxol on contraction, Ca2+ tran-sient and L-type Ca2+ current in rat ventricular myocytes. J Physiol1999;516:409–19.

[12] Howarth FC, Qureshi MA, Bracken NK, Winlow W, Singh J.Time-dependent effects of streptozotocin-induced diabetes on con-traction of ventricular myocytes from rat heart. Emirates Med J2001;19:35–41.

[13] Howarth FC, Qureshi MA, White E. Effects of hyperosmotic shrink-ing on ventricular myocyte shortening and intracellular Ca(2+) instreptozotocin-induced diabetic rats. Pflugers Arch 2002;444:446–51.

[14] Korytkowski MT, Ladenson PW. Age-related changes in basal andsodium-stimulated atrial and plasma atrial natriuretic factor (ANF)in the rat. J Gerontol 1991;46:B107–10.

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nd ventricular myocytes and more specifically the effecNP on the Ca2+ transient may form part of a physiologicrotective response to deal with defects in the heart tha

requently associated with diabetes including fibrosis, hyrophy and altered contractile function.

cknowledgements

This study was supported by an Interdisciplinary Grom the United Arab Emirates University, Al Ain, U.A.E

travel bursary from the British Council, Abu Dhabi ahe British Heart Foundation. The technical support ofar Qureshi, Dr. Irwin Chandranath, Dr. Sheela Benedicamad Ponery was highly appreciated.

eferences

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