3 RESULTS
3.5 Biological activity of BNP1-32 and BNP metabolites in vitro
3.5.1 Natriuretic peptide receptor-transfected HEK293 cells
3.5.1.1 Functionality of the receptors
HEK293 cells have been transfected with the receptors NPRA or NPRB. First of all, the transfection and functional expression of the receptors was proven by stimulating the transfected cells with ANP, BNP1-32, and CNP and determining the cGMP generation in comparison to that of cell culture medium (DMEM)-stimulated cells, which served as controls.
Figure 39 shows the quantification of the cGMP amount generated by the stimulation of such transfected cells. Stimulation of NPRA-transfected HEK293 cells with ANP and BNP1-32 led to the generation of about 300 pmol/ml cGMP (Figure 39a). Stimulation
Figure 39: Quantification of cGMP levels after stimulation of a) NPRA- and b) NPRB-transfected HEK293 cells with cell culture medium (control), or ANP, BNP1-32 or CNP with a peptide concentration of 10-7 M. Significances were calculated with One-way ANOVA: *** P < 0.001 vs. control [n = 3, in triplicates].
NPRA-transfected HEK293 cells
control ANP BNP1-32 CNP
0 100 200
300 ***
***
a
cGMP release (pmol/ml)
NPRB-transfected HEK293 cells
control ANP BNP1-32 CNP
0 100 200
300 ***
cGMP release (pmol/ml)
b
with CNP did not lead to cGMP formation, since CNP only has a marginal affinity towards NPRA, but has a high affinity towards the NPRB (see Introduction section 1.1.3). Thus, conversely, CNP stimulation of NPRB-transfected HEK293 cells led to the generation of about 250 pmol/ml cGMP, whereas ANP and BNP1-32 stimulation did not lead to a noteworthy generation of the second messenger (Figure 39b).
3.5.1.2 Stimulation of NPRA-transfected HEK293 cells
Since functionality of the receptors has been proven, NPRA-transfected HEK293 cells have been stimulated with BNP1-32 and BNP metabolites. Figure 40 summarizes the amount of cGMP generated after such stimulation.
All metabolites were able to stimulate significant cGMP generation compared to the control confirming the hypothesis that, although the new BNP metabolites are truncated on different positions, they are still bioactive.
Notably, only the renal metabolite BNP7-32 was less active compared to BNP1-32, while all other metabolites showed similar activities or were even more efficient in stimulating cGMP formation. In fact, the C-terminally truncated metabolites BNP1-30 and BNP1-29 were more bioactive compared to BNP1-32. The shortest metabolite BNP7-30 was in contrast to the N-terminally truncated peptide BNP7-32 similarly bioactive compared to BNP1-32. Obviously, the negative influence of an N-terminal
Figure 40: Quantification of cGMP levels after stimulation of NPRA-transfected HEK293 cells with cell culture medium (control), BNP1-32, BNP1-30, BNP1-29, BNP7-32, and BNP7-30 with a peptide concentration of 10-7 M. Significances were calculated with One-way ANOVA: *** P < 0.001 vs. control;
# P < 0.05; ## P < 0.01; ### P < 0.001 vs. BNP1-32 [n = 4, in triplicates].
NPRA-transfected HEK293 cells
control
BNP1-32
BNP1-30
BNP1-29
BNP7-32
BNP7-30 0
100 200 300
400 #
##
###
***
***
***
***
***
cGMP release (pmol/ml)
truncation like in BNP7-32 seems to be buffered by an additional C-terminal truncation in BNP7-30.
3.5.1.3 Stimulation of NPRB-transfected HEK293 cells
It was demonstrated before that BNP1-32 does stimulate NPRA but not NPRB (Figure 39). To investigate if the C- and N-terminal truncations of the peptide do influence receptor specificity, NPRB-single-transfected HEK293 cells have been stimulated (Figure 41).
As stated before, BNP1-32 is only marginally able to stimulate cGMP generation in comparison to CNP. As seen in Figure 41, the stimulation with the peptide led to the generation of approx. 3 pmol/ml cGMP. Surprisingly, BNP1-30 showed much higher effects on NPRB-transfected cells compared to BNP1-32. Although cGMP levels were still low compared to those arising from NPRA-stimulation, the amount of cGMP generated is tripled in comparison to usual levels achieved by stimulation with BNP1-32. Notably, other metabolites did not show this increased effect on NPRB.
3.5.1.4 Stimulation of NPRA & NPRB double-transfected HEK293 cells Since the experiments on the NPRB revealed that BNP1-30 stimulation led to a significantly higher cGMP generation compared to BNP1-32, it was tested whether the stimulation of both receptors in parallel would lead to an additional increase in
Figure 41: cGMP levels after stimulation of NPRB-transfected HEK293 cells with cell culture medium (control), BNP1-32, and BNP metabolites with a peptide concentration of 10-7 M. Significances were calculated with One-way ANOVA: # P < 0.05; *** P < 0.001 vs. BNP1-32 [n = 4, in triplicates].
NPRB-transfected HEK293 cells
control
BNP1-32
BNP1-30
BNP1-29
BNP7-32
BNP7-30 0
2 4 6 8 10 12
14 ###
*** ***
***
***
***
cGMP release (pmol/ml)
cGMP formation upon stimulation with BNP1-30. Thus, to complete the investigations, the bioactivity of BNP1-32 and the BNP metabolites was investigated in NPRA&B-double-transfected HEK293 cells.
As shown in Figure 42a, all metabolites were able to stimulate significant cGMP generation compared to the control. The graph shows the same pattern as for the NPRA-single transfected cells, since this receptor is the major effector for cGMP formation by BNPs. Notably, cGMP values were lower in comparison to those measured in NPRA-single transfected cells, although same plasmid concentration was used in both approaches. Very recently, it was discovered in our working group, that subunits of NPRA and NPRB can interact with each other forming heterodimers with altered ligand affinities263.Thus, cGMP values from single-transfected cells might not necessarily summarize. To provide insight into the potency of the peptides, experiments with different concentrations of the peptides have been made and dose response curves have been calculated. Figure 42b shows the different curves for each metabolite applied. As revealed before, BNP1-29 showed highest efficacy with a top value of 86 pmol/ml, whereas BNP7-32 had the lowest with a top value of
Figure 42: a) Quantification of the cGMP levels after stimulation of NPRA & B-cotransfected HEK293 cells with BNP1-32, BNP1-30, BNP1-29, BNP7-32, and BNP7-30 with a peptide concentration of 10-7 M .Significances were calculated with One-way ANOVA: *** P < 0.001 vs. control; ## P < 0.01; ### P < 0.001 vs. BNP1-32; §§ P < 0.01 vs. BNP1-30 [n = 3 in triplicates] b) dose response curves obtained from the stimulation of NPRA & B-cotransfected HEK293 cells with BNP1-32, BNP1-30, BNP1-29, BNP7-32, and BNP7-30. Significances were calculated with Two-way ANOVA: ###P < 0.001 vs. BNP1-32 (although not indicated, all curves are *** P < 0.001 vs. control) [n = 3 in triplicates].
NPRA- & B-cotransfected HEK293 cells
control
BNP1-32
BNP1-30
BNP1-29
BNP7-32
BNP7-30 0
20 40 60 80 100
##
###
a
***
***
***
***
***
§§
cGMP release (pmol/ml)
Dose response curve
NPRA- & B-cotransfected HEK293 cells
-9 -8 -7 -6
0 20 40 60 80 100
BNP1-29 BNP1-30 BNP1-32
BNP7-32 BNP7-30 control
###
b
log [peptide], log M
cGMP release (pmol/ml)
47 pmol/ml with a peptide concentration of 5 x 10-7 M (Table 16). The EC50 values were similar for all peptides (also Table 16). Significance towards BNP1-32 was reached only for BNP1-29 using Two-way ANOVA caused by the high number of groups being compared. In a comparison using only one curve in comparison to that of BNP1-32, BNP1-30 and BNP7-30 are also significantly different to BNP1-32.
Table 16: Summary of EC50 and Top values from dose response curve.
Besides using receptor-transfected cells, bioactivity of the BNP metabolites was also tested using cells endogenously expressing natriuretic peptide receptors. Cell types, which are known to be essential in blood pressure homeostasis, have been utilized for such investigations. Results are described in the next chapter.